Draft genome sequence of three hydrocarbon-degrading Pseudomonadota strains isolated from an abandoned century-old oil exploration well.

We present genome sequences of three Pseudomonadota strains isolated from an abandoned century-old oil exploration well. A Pseudomonas sp. genome showed a size of 5,378,420 bp, while Acinetobacter genomes sized 3,522,593 and 3,864,311 bp. Genomes included catabolic genes for benzoate, 4-hydroxybenzoate, salicylate, vanillate, indoleacetate, anthranilate, n-alkanes, 4-hydroxyphenylacetate, phenylacetate, among others.

Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds.

Stutzerimonas balearica (Pseudomonas balearica) has been found principally in oil-polluted environments. The capability of S. balearica to thrive from the degradation of pollutant compounds makes it a species of interest for potential bioremediation applications. However, little has been reported about the diversity of S. balearica. In this study, genome sequences of S. balearica strains from different origins were analyzed, revealing that it is a diverse species with an open pan-genome that will continue revealing new genes and functionalities as the genomes of more strains are sequenced. The nucleotide signatures and intra- and inter-species variation of the 16S rRNA genes of S. balearica were reevaluated. A strategy of screening 16S rRNA gene sequences in public databases enabled the detection of 158 additional strains, of which only 23% were described as S. balearica. The species was detected from a wide range of environments, although mostly from aquatic and polluted environments, predominantly related to petroleum oil. Genomic and phenotypic analyses confirmed that S. balearica possesses varied inherent capabilities for aromatic compounds degradation. This study increases the knowledge of the biology and diversity of S. balearica and will serve as a basis for future work with the species.

Methylotrophs and Hydrocarbon-Degrading Bacteria Are Key Players in the Microbial Community of an Abandoned Century-Old Oil Exploration Well.

In this work, we studied the microbial community and the physicochemical conditions prevailing in an exploratory oil well, abandoned a century ago, located in the Cahuita National Park (Costa Rica). According to our analysis, Cahuita well is characterized by a continuous efflux of methane and the presence of a mixture of hydrocarbons including phenanthrene/anthracene, fluoranthene, pyrene, dibenzothiophene, tricyclic terpanes, pyrene, sesquiterpenes, sterane, and n-alkanes. Based on the analysis of 16S rRNA gene amplicons, we detected a significant abundance of methylotrophic bacteria such as Methylobacillus (6.3-26.0% of total reads) and Methylococcus (4.1-30.6%) and the presence of common genera associated with hydrocarbon degradation, such as Comamonas (0.8-4.6%), Hydrogenophaga (1.5-3.3%) Rhodobacter (1.0-4.9%), and Flavobacterium (1.1-6.5%). The importance of C1 metabolism in this niche was confirmed by amplifying the methane monooxygenase (MMO)-encoding gene (pmo) from environmental DNA and the isolation of two strains closely related to Methylorubrum rhodesianum and Paracoccus communis with the ability to growth using methanol and formate as sole carbon source respectively. In addition, we were able to isolated 20 bacterial strains from the genera Pseudomonas, Acinetobacter, and Microbacterium which showed the capability to grow using the hydrocarbons detected in the oil well as sole carbon source. This work describes the physicochemical properties and microbiota of an environment exposed to hydrocarbons for 100 years, and it not only represents a contribution to the understanding of microbial communities in environments with permanently high concentrations of these compounds but also has biotechnological implications for bioremediation of petroleum-polluted sites.

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids.

Rieske-type two-component vanillate O-demethylases (VanODs) catalyze conversion of the lignin-derived monomer vanillate into protocatechuate in several bacterial species. Currently, VanODs have received attention because of the demand of effective lignin valorization technologies, since these enzymes own the potential to catalyze methoxy group demethylation of distinct lignin monomers. In this work, we identified a phylogenetically divergent VanOD from Rhodococcus ruber R1, only distantly related to previously described homologues and whose presence, along with a 3-hydroxybenzoate/gentisate pathway, correlated with the ability to grow on other meta-methoxylated aromatics, such as 3-methoxybenzoate and 5-methoxysalicylate. The complementation of catabolic abilities by heterologous expression in a host strain unable to grow on vanillate, and subsequent resting cell assays, suggest that the vanAB genes of R1 strain encode a proficient VanOD acting on different vanillate-like substrates; and also revealed that a methoxy group in the meta position and a carboxylic acid moiety in the aromatic ring are key for substrate recognition. Phylogenetic analysis of the oxygenase subunit of bacterial VanODs revealed three divergent groups constituted by homologues found in Proteobacteria (Type I), Actinobacteria (Type II), or Proteobacteria/Actinobacteria (Type III) in which the R1 VanOD is placed. These results suggest that VanOD from R1 strain, and its type III homologues, expand the range of methoxylated aromatics used as substrates by bacteria.

Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species.

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner-Doudoroff (ED), pentose-phosphate (PP), and lower Embden-Meyerhoff-Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via ß-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway.

The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a ß-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α-, ß- and γ-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self-sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes.

Widespread distribution of hmf genes in Proteobacteria reveals key enzymes for 5-hydroxymethylfurfural conversion.

Furans represent a class of promising chemicals, since they constitute valuable intermediates in conversion of biomass into sustainable products intended to replace petroleum-derivatives. Conversely, generation of furfural and 5-hydroxymethylfurfural (HMF) as by-products in lignocellulosic hydrolysates is undesirable due its inhibitory effect over fermentative microorganisms. Therefore, the search for furans-metabolizing bacteria has gained increasing attention since they are valuable tools to solve these challenging issues. A few bacterial species have been described at genetic level, leading to a proposed HMF pathway encoded by a set of genes termed hmf/psf, although some enzymatic functions are still elusive. In this work we performed a genomic analysis of major subunits of furoyl-CoA dehydrogenase orthologues, revealing that the furoic acid catabolic route, key intermediate in HMF biodegradation, is widespread in proteobacterial species. Additionally, presence/absence profiles of hmf/psf genes in selected proteobacterial strains suggest parallel and/or complementary roles of enzymes with previously unclear function that could be key in HMF conversion. The furans utilization pattern of selected strains harboring different hmf/psf gene sets provided additional support for bioinformatic predictions of the relevance of some enzymes. On the other hand, at least three different types of transporter systems are clustered with hmf/psf genes, whose presence is mutually exclusive, suggesting a core and parallel role in furans transport in Proteobacteria. This study expands the number of bacteria that could be recruited in biotechnological processes for furans biodetoxification and predicts a core set of genes required to establish a functional HMF pathway in heterologous hosts for metabolic engineering endeavors.

Complete Multipartite Genome Sequence of the Cupriavidus basilensis Type Strain, a 2,6-Dichlorophenol-Degrading Bacterium.

We report the complete 8.94-Mb genome sequence of the type strain of Cupriavidus basilensis (DSM 11853T = CCUG 49340T = RK1T), formed by two chromosomes and six putative plasmids, which offers insights into its chloroaromatic-biodegrading capabilities.

Transcriptional control of 2,4-dinitrotoluene degradation in Burkholderia sp. R34 bears a regulatory patch that eases pathway evolution.

The dnt pathway of Burkholderia sp. R34 is in the midst of an evolutionary journey from its ancestral, natural substrate (naphthalene) towards a new xenobiotic one [2,4-dinitrotoluene (DNT)]. The gene cluster encoding the leading multicomponent ring dioxygenase (DntA) has activity on the old and the new substrate, but it is induced by neither. Instead, the transcriptional factor encoded by the adjacent gene (dntR) activates expression of the dnt cluster upon addition of salicylate, one degradation intermediate of the ancestral naphthalene route but not any longer a substrate/product of the evolved DntA enzyme. Fluorescence of cells bearing dntA-gfp fusions revealed that induction of the dnt genes by salicylate was enhanced upon exposure to bona fide DntA substrates, i.e., naphthalene or DNT. Such amplification was dependent on effective dioxygenation of these pathway-specific head compounds, which thereby fostered expression of the cognate catabolic operon. The phenomenon seems to happen not through direct binding to a cognate transcriptional factor but through the interplay of a non-specific regulator with a substrate-specific enzyme. This regulatory scenario may ease transition of complete catabolic operons (i.e. enzymes plus regulatory devices) from one substrate to another without loss of fitness during the evolutionary roadmap between two optimal specificities.

The faulty SOS response of Pseudomonas putida KT2440 stems from an inefficient RecA-LexA interplay.

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

Complete Genome Sequence of Rhodococcus ruber R1, a Novel Strain Showing a Broad Catabolic Potential toward Lignin-Derived Aromatics.

Rhodococcus ruber R1 was isolated from a pulp mill wastewater treatment plant because of its ability to use methoxylated aromatics as growth substrates. We report the 5.56-Mb genome sequence of strain R1, which can provide insights into the biodegradation of lignin-derived phenolic monomers and potentially support processes for lignocellulose conversion.

Draft Genome Sequences of Two Pseudomonas Strains That Are Able To Use Furan Derivatives as Their Sole Carbon Source.

Pseudomonas sp. strains ALS1279 and ALS1131 were isolated from wastewater treatment facilities on the basis of their ability to use furfural, a key lignocellulose-derived inhibitor, as their only carbon source. Here, we present the draft genome sequences of both strains, which can shed light on catabolic pathways for furan compounds in pseudomonads.

Evolving metabolism of 2,4-dinitrotoluene triggers SOS-independent diversification of host cells.

The molecular mechanisms behind the mutagenic effect of reactive oxygen species (ROS) released by defective metabolization of xenobiotic 2,4-dinitrotoluene (DNT) by a still-evolving degradation pathway were studied. To this end, the genes required for biodegradation of DNT from Burkholderia cepacia R34 were implanted in Escherichia coli and the effect of catabolizing the nitroaromatic compound monitored with stress-related markers and reporters. Such a proxy of the naturally-occurring scenario faithfully recreated the known accumulation of ROS caused by faulty metabolism of DNT and the ensuing onset of an intense mutagenesis regime. While ROS triggered an oxidative stress response, neither homologous recombination was stimulated nor the recA promoter activity increased during DNT catabolism. Analysis of single-nucleotide changes occurring in rpoB during DNT degradation suggested a relaxation of DNA replication fidelity rather than direct damage to DNA. Mutants frequencies were determined in strains defective in either converting DNA damage into mutagenesis or mediating inhibition of mismatch repair through a general stress response. The results revealed that the mutagenic effect of ROS was largely SOS-independent and stemmed instead from stress-induced changes of rpoS functionality. Evolution of novel metabolic properties thus resembles the way sublethal antibiotic concentrations stimulate the appearance of novel resistance genes.

Draft genome sequences of Cylindrospermopsis raciborskii strains CS-508 and MVCC14, isolated from freshwater bloom events in Australia and Uruguay.

Members of the genus Cylindrospermopsis represent an important environmental and health concern. Strains CS-508 and MVCC14 of C. raciborskii were isolated from freshwater reservoirs located in Australia and Uruguay, respectively. While CS-508 has been reported as non-toxic, MVCC14 is a saxitoxin (STX) producer. We annotated the draft genomes of these C. raciborskii strains using the assembly of reads obtained from Illumina MiSeq sequencing. The final assemblies resulted in genome sizes close to 3.6 Mbp for both strains and included 3202 ORFs for CS-508 (in 163 contigs) and 3560 ORFs for MVCC14 (in 99 contigs). Finally, both the average nucleotide identity (ANI) and the similarity of gene content indicate that these two genomes should be considered as strains of the C. raciborskii species.

Diurnal Changes in Active Carbon and Nitrogen Pathways Along the Temperature Gradient in Porcelana Hot Spring Microbial Mat.

Composition, carbon and nitrogen uptake, and gene transcription of microbial mat communities in Porcelana neutral hot spring (Northern Chilean Patagonia) were analyzed using metagenomics, metatranscriptomics and isotopically labeled carbon (H13CO3) and nitrogen (15NH4Cl and K15NO3) assimilation rates. The microbial mat community included 31 phyla, of which only Cyanobacteria and Chloroflexi were dominant. At 58°C both phyla co-occurred, with similar contributions in relative abundances in metagenomes and total transcriptional activity. At 66°C, filamentous anoxygenic phototrophic Chloroflexi were >90% responsible for the total transcriptional activity recovered, while Cyanobacteria contributed most metagenomics and metatranscriptomics reads at 48°C. According to such reads, phototrophy was carried out both through oxygenic photosynthesis by Cyanobacteria (mostly Mastigocladus) and anoxygenic phototrophy due mainly to Chloroflexi. Inorganic carbon assimilation through the Calvin-Benson cycle was almost exclusively due to Mastigocladus, which was the main primary producer at lower temperatures. Two other CO2 fixation pathways were active at certain times and temperatures as indicated by transcripts: 3-hydroxypropionate (3-HP) bi-cycle due to Chloroflexi and 3-hydroxypropionate-4-hydroxybutyrate (HH) cycle carried out by Thaumarchaeota. The active transcription of the genes involved in these C-fixation pathways correlated with high in situ determined carbon fixation rates. In situ measurements of ammonia assimilation and nitrogen fixation (exclusively attributed to Cyanobacteria and mostly to Mastigocladus sp.) showed these were the most important nitrogen acquisition pathways at 58 and 48°C. At 66°C ammonia oxidation genes were actively transcribed (mostly due to Thaumarchaeota). Reads indicated that denitrification was present as a nitrogen sink at all temperatures and that dissimilatory nitrate reduction to ammonia (DNRA) contributed very little. The combination of metagenomic and metatranscriptomic analysis with in situ assimilation rates, allowed the reconstruction of day and night carbon and nitrogen assimilation pathways together with the contribution of keystone microorganisms in this natural hot spring microbial mat.

The interplay of EIIANtr with C-source regulation of the Pu promoter of Pseudomonas putida mt-2.

The presence of some sugars (e.g. glucose) downregulates the activity of the Pu promoter of plasmid pWW0 of Pseudomonas putida mt-2, which drives the upper TOL operon for biodegradation of m-xylene. Genetic evidence produced 20 years ago documented an effect of the EIIANtr (PtsN) protein of the nitrogen-related phosphoenolpyruvate-dependent phosphotransferase system (PTSNtr ) in such a C-source control of Pu activity. In this study, we have exploited the wealth of recent information on the PTS of P. putida as well as transcriptomic data available in the last few years on this bacterium to revisit this question - and the role of EIIANtr as such. To this end, we examined Pu output under physiological conditions known to either phosphorylate PTS proteins to saturation or to deplete them altogether from high-energy phosphate. The results showed that Pu activity is checked by EIIANtr regardless of its phosphorylation state. However, such inhibition is intensified during growth on glucose (which correlates with more phosphate-free EIIANtr ) and partially relieved in fructose, which triggers phosphorylation of PTS proteins. These data explain former inconsistencies on the Pu-PTSNtr interplay and provides a better understanding of the metabolic and regulatory retroactivity between the TOL plasmid and its host metabolism.

The Metabolic Redox Regime of Pseudomonas putida Tunes Its Evolvability toward Novel Xenobiotic Substrates.

During evolution of biodegradation pathways for xenobiotic compounds involving Rieske nonheme iron oxygenases, the transition toward novel substrates is frequently associated with faulty reactions. Such events release reactive oxygen species (ROS), which are endowed with high mutagenic potential. In this study, we evaluated how the operation of the background metabolic network by an environmental bacterium may either foster or curtail the still-evolving pathway for 2,4-dinitrotoluene (2,4-DNT) catabolism. To this end, the genetically tractable strain Pseudomonas putida EM173 was implanted with the whole genetic complement necessary for the complete biodegradation of 2,4-DNT (recruited from the environmental isolate Burkholderia sp. R34). By using reporter technology and direct measurements of ROS formation, we observed that the engineered P. putida strain experienced oxidative stress when catabolizing the nitroaromatic substrate. However, the formation of ROS was neither translated into significant activation of the SOS response to DNA damage nor did it result in a mutagenic regime (unlike what has been observed in Burkholderia sp. R34, the original host of the pathway). To inspect whether the tolerance of P. putida to oxidative challenges could be traced to its characteristic reductive redox regime, we artificially altered the NAD(P)H pool by means of a water-forming, NADH-specific oxidase. Under the resulting low-NAD(P)H status, catabolism of 2,4-DNT triggered a conspicuous mutagenic and genomic diversification scenario. These results indicate that the background biochemical network of environmental bacteria ultimately determines the evolvability of metabolic pathways. Moreover, the data explain the efficacy of some bacteria (e.g., pseudomonads) to host and evolve with new catabolic routes.IMPORTANCE Some environmental bacteria evolve with new capacities for the aerobic biodegradation of chemical pollutants by adapting preexisting redox reactions to novel compounds. The process typically starts by cooption of enzymes from an available route to act on the chemical structure of the substrate-to-be. The critical bottleneck is generally the first biochemical step, and most of the selective pressure operates on reshaping the initial reaction. The interim uncoupling of the novel substrate to preexisting Rieske nonheme iron oxygenases usually results in formation of highly mutagenic ROS. In this work, we demonstrate that the background metabolic regime of the bacterium that hosts an evolving catabolic pathway (e.g., biodegradation of the xenobiotic 2,4-DNT) determines whether the cells either adopt a genetic diversification regime or a robust ROS-tolerant status. Furthermore, our results offer new perspectives to the rational design of efficient whole-cell biocatalysts, which are pursued in contemporary metabolic engineering.

Genomic features of "Candidatus Venteria ishoeyi", a new sulfur-oxidizing macrobacterium from the Humboldt Sulfuretum off Chile.

The Humboldt Sulfuretum (HS), in the productive Humboldt Eastern Boundary Current Upwelling Ecosystem, extends under the hypoxic waters of the Peru-Chile Undercurrent (ca. 6°S and ca. 36°S). Studies show that primeval sulfuretums held diverse prokaryotic life, and, while rare today, still sustain species-rich giant sulfur-oxidizing bacterial communities. We here present the genomic features of a new bacteria of the HS, "Candidatus Venteria ishoeyi" ("Ca. V. ishoeyi") in the family Thiotrichaceae.Three identical filaments were micro-manipulated from reduced sediments collected off central Chile; their DNA was extracted, amplified, and sequenced by a Roche 454 GS FLX platform. Using three sequenced libraries and through de novo genome assembly, a draft genome of 5.7 Mbp, 495 scaffolds, and a N50 of 70 kbp, was obtained. The 16S rRNA gene phylogenetic analysis showed that "Ca. V. ishoeyi" is related to non-vacuolate forms presently known as Beggiatoa or Beggiatoa-like forms. The complete set of genes involved in respiratory nitrate-reduction to dinitrogen was identified in "Ca. V. ishoeyi"; including genes likely leading to ammonification. As expected, the sulfur-oxidation pathway reported for other sulfur-oxidizing bacteria were deduced and also, key inorganic and organic carbon acquisition related genes were identified. Unexpectedly, the genome of "Ca. V. ishoeyi" contained numerous CRISPR repeats and an I-F CRISPR-Cas type system gene coding array. Findings further show that, as a member of an eons-old marine ecosystem, "Ca. V. ishoeyi" contains the needed metabolic plasticity for life in an increasingly oxygenated and variable ocean.

Cupriavidus pinatubonensis AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid.

BACKGROUND: Microbial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure. RESULTS: Exposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells. CONCLUSIONS: The current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs.