Background: Copper mining has led to Cu pollution in agricultural soils. In this report, the effects of Cu pollution on bacterial communities of agricultural soils from Valparaiso region, central Chile, were studied. Denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes was used for the characterization of bacterial communities from Cu-polluted and non-polluted soils. Cu-resistant bacterial strains were isolated from Cu-polluted soils and characterized. Results: DGGE showed a similar high number of bands and banding pattern of the bacterial communities from Cu-polluted and non-polluted soils. The presence of copA genes encoding the multi-copper oxidase that confers Cu-resistance in bacteria was detected by PCR in metagenomic DNA from the three Cu-polluted soils, but not in the non-polluted soil. The number of Cu-tolerant heterotrophic cultivable bacteria was significantly higher in Cu-polluted soils than in the non-polluted soil. Ninety two Cu-resistant bacterial strains were isolated from three Cu-polluted agricultural soils. Five isolated strains showed high resistance to copper (MIC ranged from 3.1 to 4.7 mM) and also resistance to other heavy metals. 16S rRNA gene sequence analyses indicate that these isolates belong to the genera Sphingomonas, Stenotrophomonas and Arthrobacter. The Sphingomonas sp. strains O12, A32 and A55 and Stenotrophomonas sp. C21 possess plasmids containing the Cu-resistance copA genes. Arthrobacter sp. O4 possesses the copA gene, but plasmids were not detected in this strain. The amino acid sequences of CopA from Sphingomonas isolates (O12, A32 and A55), Stenotrophomonas strain (C21) and Arthrobacter strain (O4) are closely related to CopA from Sphingomonas, Stenotrophomonas and Arthrobacter strains, respectively. Conclusions: This study suggests that bacterial communities of agricultural soils from central Chile exposed to long-term Cu-pollution have been adapted by acquiring Cu genetic determinants. Five bacterial isolates showed high copper resistance and additional resistance to other heavy metals. Detection of copA gene in plasmids of four Cu-resistant isolates indicates that mobile genetic elements are involved in the spreading of Cu genetic determinants in polluted environments.

Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg²⁺. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg²⁺, 0.12 and CH₃Hg⁺, 0.08. The addition of Hg²⁺ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg²⁺ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg²⁺ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg²⁺ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg²⁺ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h. A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).

For the past three decades, Cupriavidus metallidurans has been one of the major model organisms for bacterial tolerance to heavy metals. Its type strain CH34 contains at least 24 gene clusters distributed over four replicons, allowing for intricate and multilayered metal responses. To gain organic mercury resistance in CH34, broad-spectrum mer genes were introduced in a previous work via conjugation of the IncP-1β plasmid pTP6. However, we recently noted that this CH34-derived strain, MSR33, unexpectedly showed an increased resistance to other metals (i.e., Co²⁺, Ni²⁺, and Cd²⁺). To thoroughly investigate this phenomenon, we resequenced the entire genome of MSR33 and compared its DNA sequence and basal gene expression profile to those of its parental strain CH34. Genome comparison identified 11 insertions or deletions (INDELs) and nine single nucleotide polymorphisms (SNPs), whereas transcriptomic analysis displayed 107 differentially expressed genes. Sequence data implicated the transposition of IS1088 in higher Co²⁺ and Ni²⁺ resistances and altered gene expression, although the precise mechanisms of the augmented Cd²⁺ resistance in MSR33 remains elusive. Our work indicates that conjugation procedures involving large complex genomes and extensive mobilomes may pose a considerable risk toward the introduction of unwanted, undocumented genetic changes. Special efforts are needed for the applied use and further development of small nonconjugative broad-host plasmid vectors, ideally involving CRISPR-related and advanced biosynthetic technologies.

Benzene, toluene, ethylbenzene and the three xylene isomers are monoaromatic contaminants widely distributed on polluted sites. Some microorganisms have developed mechanisms to degrade these compounds, but their aerobic and anaerobic degradation is inhibited in presence of heavy metals, such as mercury or lead. In this report, the degradation of benzene and other aromatic compounds catalyzed by the metal resistant bacterium Cupriavidus metallidurans CH34 was characterized. A metabolic reconstruction of aromatic catabolic pathways was performed based on bioinformatics analyses. Functionality of the predicted pathways was confirmed by growing strain CH34 on benzene, toluene, o- xylene, p- cymene, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, homogentisate, catechol, naphthalene, and 2-aminophenol as sole carbon and energy sources. Benzene catabolic pathway was further characterized. Results showed that firstly benzene is transformed into phenol and, thereafter, into catechol. Benzene is degraded under aerobic conditions via a combined pathway catalyzed by three Bacterial Multicomponent Monooxygenases: a toluene-2-monoxygenase (TomA012345), a toluene-4-monooxygenase (TmoABCDEF) and a phenol-2-hydroxylase (PhyZABCDE). A catechol-2,3-dioxygenase (TomB) expressed at early exponential phase cleaves the catechol ring in meta -position; an ortho -cleavage of catechol is accomplished by a catechol-1,2-dioxygenase (CatA) at late exponential phase instead. This study additionally shows that C. metallidurans CH34 is capable of degrading benzene in presence of heavy metals, such as Hg(II) or Pb(II). This capability of degrading aromatic compounds in presence of heavy metals is rather unusual among environmental bacteria; therefore, C. metallidurans CH34 seems to be a promising candidate for developing novel bioremediation process for multi-contaminated environments. HIGHLIGHTS: The strain Cupriavidus metallidurans CH34 is capable to degrade benzene aerobically Benzene oxydation is mediated by bacterial multicomponent monoxygenases Strain CH34 is able to grow using a broad range of aromatic compounds as sole carbon and energy source Benzene degradation occurs even in presence of heavy metals such as mercury and lead

Osorno volcano (41.1° S, 72° W) is located in the Andean Southern Volcanic Zone. The volcano lies within a national park as part of the protected areas system. This setting provides an opportunity to compare soil microbial communities between sectors with (H) and without (NI) anthropogenic activities within a volcanic territory. To do so, we selected one of the most visited volcanoes in Chilean Patagonia to examine composition, diversity (taxonomic and phylogenetic), and co-presence and mutual exclusion interaction networks between members of volcanic soil bacterial communities. Soil DNA was extracted, and the 16S rRNA gene was analyzed by high-throughput DNA sequencing, followed by taxonomic identification. The most prevalent phylum across all sites (H and NI) was Pseudomonadota, followed by Acidobacteriota, Actinobacteriota, and Chloroflexota. Based on taxonomic and phylogenetic indices, we found that the diversity of bacteria was significantly less in the humanized area than in the non-intervened areas. Beta diversity analysis also revealed a clear separation between humanized and non-intervened soils. Additionally, a decrease in network connectivity was observed at NI sites. Our results provide clear evidence that anthropogenic factors, such as tourism, vehicle parking, and combustion processes, are key drivers shaping bacterial community structure in volcanic soils, with potential consequences for ecosystem health and the capacity to provide ecosystem services.