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Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

ABSTRACT: The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoR^Ac), an organism used in the bioleaching process of copper ores. CsoR^Ac possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR^Ac monomer in vitro combined with atomic absorption spectroscopy analysis. Cu^I-CsoR^Ac and apo-CsoR^Ac share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (K_D = 2.26 × 10^-18 M and 0.53 × 10^-15 M) and Cu(I) binding affinity of apo-CsoR^Ac were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR^Ac did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoR^Ac. Moreover, apo-CsoR^Ac could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoR^Ac, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

SUBMITTER: Hou S

PROVIDER: S-EPMC8315176 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acidithiobacillus caldus is an extremely acidophilic sulfur-oxidizer with specialized characteristics, such as tolerance to low pH and heavy metal resistance. To gain novel insights into its genetic complexity, we chosen six A. caldus strains for comparative survey. All strains analyzed in this study differ in geographic origins as well as in ecological preferences. Based on phylogenomic analysis, we clustered the six A. caldus strains isolated from various ecological niches into two groups: group 1 strains with smaller genomes and group 2 strains with larger genomes. We found no obvious intraspecific divergence with respect to predicted genes that are related to central metabolism and stress management strategies between these two groups. Although numerous highly homogeneous genes were observed, high genetic diversity was also detected. Preliminary inspection provided a first glimpse of the potential correlation between intraspecific diversity at the genome level and environmental variation, especially geochemical conditions. Evolutionary genetic analyses further showed evidence that the difference in environmental conditions might be a crucial factor to drive the divergent evolution of A. caldus species. We identified a diverse pool of mobile genetic elements including insertion sequences and genomic islands, which suggests a high frequency of genetic exchange in these harsh habitats. Comprehensive analysis revealed that gene gains and losses were both dominant evolutionary forces that directed the genomic diversification of A. caldus species. For instance, horizontal gene transfer and gene duplication events in group 2 strains might contribute to an increase in microbial DNA content and novel functions. Moreover, genomes undergo extensive changes in group 1 strains such as removal of potential non-functional DNA, which results in the formation of compact and streamlined genomes. Taken together, the findings presented herein show highly frequent gene turnover of A. caldus species that inhabit extremely acidic environments, and shed new light on the contribution of gene turnover to the evolutionary adaptation of acidophiles.

Project description:BackgroundAcidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.Principal findingsComparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions.SignificanceFor many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.

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Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

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