Isolation and Characterization of Copper Tolerant Bacterial Species Habituating in Copper Mines and Study of Their Potentiality as a Plant Growth Stimulator

Copper (Cu) is a vital micronutrient for all living organisms below its toxicity limit. Various industrial activities, mining deposits, excessive use of harmful chemicals, waste discharges, and drugs are the main reason for the emerging copper concentration. Emphases of the current study were to isolate and characterize highly copper-tolerating bacterial (CTB) species from a copper-contaminated site. In enrichment culture techniques, 24 copper tolerant microbial isolates were evaluated and the maximum tolerable concentration (MTC) was determined using various concentrations of copper sulfate pentahydrate (CuSO4.5H2O) solution. Three bacterial strains named GKSM2, GKSM6, and GKSM11 were tolerant to 350 mg/l of CuSO4.5H2O. The 16S rRNA gene sequencing and phylogeny data revealed that these CTB belong to species Bacillus zanthoxyli, Bacillus stercoris, and Pseudomonas alcaliphila species. CTB showed their optimized growth at moderate salt concentration (0.1-0.5M NaCl), temperature range (20-45˚C) and wide pH range (pH 5.0-11). All the strains can produce various Plant growth stimulating (PGS) traits viz., phytohormones (IAA, GA), proline, nitrogen fixation, ammonification, and antioxidant enzymes in presence and absence of Cu2+ stress. The result displays that adsorption of Cu2+ ions evidenced by TEM, SEM, and SEM-EDX analysis.

Gene Expression Analysis of Three Putative Copper-Transporting ATPases in Copper-Tolerant Fibroporia radiculosa

Copper tolerance of brown-rot basidiomycete decay fungi can lessen the efficacy of copper-containing wood preservatives for wood products in-service. The purpose of this study was to evaluate wood mass loss and differential expression of three genes that have putative annotations for copper-transporting ATPase pumps (FIBRA_00974, FIBRA_04716, and FIBRA_01430). Untreated southern pine (SP) and SP treated with three concentrations of ammoniacal copper citrate (CC, 0.6, 1.2, and 2.4%) were exposed to two copper-tolerant Fibroporia radiculosa isolates (FP-90848-T and L-9414-SP) and copper-sensitive Gloeophyllum trabeum isolate (MAD 617) in a 4-week-long standard decay test (AWPA E10-19). Decay of copper-treated wood was inhibited by G. trabeum (p = 0.001); however, there was no inhibition of decay with increasing copper concentrations by both F. radiculosa isolates. Initially, G. trabeum and one F. radiculosa isolate (L-9414-SP) highly upregulated FIBRA_00974 and FIBRA_04716 on copper-treated wood at week 1 (p = 0.005), but subsequent expression was either not detected or was similar to expression on untreated wood (p = 0.471). The other F. radiculosa isolate (FP-90848-T) downregulated FIBRA_00974 (p = 0.301) and FIBRA_04716 (p = 0.004) on copper-treated wood. FIBRA_01430 expression by G. trabeum was not detected, but was upregulated by both F. radiculosa FP-90848-T (p = 0.481) and L-9414-SP (p = 0.392). Results from this study suggest that all three test fungi utilized different mechanisms when decaying copper-treated wood. Additionally, results from this study do not provide support for the involvement of these putative gene annotations for copper-transporting ATPase pumps in the mechanism of copper-tolerance.

Essential Gene Clusters Involved in Copper Tolerance Identified in Acinetobacter baumannii Clinical and Environmental Isolates

Copper is widely used as antimicrobial in agriculture and medicine. Copper tolerance mechanisms of pathogenic bacteria have been proven to be required for both copper tolerance and survival during bacterial infections. Here, we determined both copper-tolerant phenotype and genotype in A. baumannii originated from clinical and environmental samples. Using copper susceptibility testing, copper-tolerant A. baumannii could be found in both clinical and environmental isolates. Genotypic study revealed that representative copper-related genes of the cluster A (cueR), B (pcoAB), and D (oprC) were detected in all isolates, while copRS of cluster C was detected in only copper-tolerant A. baumannii isolates. Moreover, we found that copper-tolerant phenotype was associated with amikacin resistance, while the presence of copRS was statistically associated with blaNDM-1. We chose the A. baumannii strain AB003 as a representative of copper-tolerant isolate to characterize the effect of copper treatment on external morphology as well as on genes responsible for copper tolerance. The morphological features and survival of A. baumannii AB003 were affected by its exposure to copper, while whole-genome sequencing and analysis showed that it carried fourteen copper-related genes located on four clusters, and cluster C of AB003 was found to be embedded on genomic island G08. Transcriptional analysis of fourteen copper-related genes identified in AB003 revealed that copper treatment induced the expressions of genes of clusters A, B, and D at the micromolar level, while genes of cluster C were over-expressed at the millimolar levels of copper. This study showed that both clinical and environmental A. baumannii isolates have the ability to tolerate copper and carried numerous copper tolerance determinants including intrinsic copper tolerance (clusters A, B, and D) and acquired copper tolerance (cluster C) that could respond to copper toxicity. Our evidence suggests that we need to reconsider the use of copper in hospitals and other medical environments to prevent the selection and spread of copper-tolerant organisms.

Bactericidal Activity of Copper-Zinc Hybrid Nanoparticles on Copper-Tolerant Xanthomonas perforans

Bacterial spot of tomato, caused by Xanthomonas perforans, X. euvesicatoria, X. vesicatoria and X. gardneri, is a major disease, contributing to significant yield losses worldwide. Over dependence of conventional copper bactericides over the last decades has led to the prevalence of copper-tolerant strains of Xanthomonas spp., making copper bactericides ineffective. Thus, there is a critical need to develop new strategies for better management of copper-tolerant Xanthomonas spp. In this study, we investigated the antimicrobial activity of a hybrid nanoparticle, copper-zinc (Cu/Zn), on copper-tolerant and sensitive strains. The hybrid nanoparticle significantly reduced bacterial growth in vitro compared to the non-treated and micron-size commercial copper controls. Tomato transplants treated with the hybrid nanoparticle had significantly reduced disease severity compared to the controls, and no phytotoxicity was observed on plants. We also studied the hybrid nanoparticle effect on the bacterial pigment xanthomonadin using Near-Infra Red Raman spectroscopy as an indicator of bacterial degradation. The hybrid nanoparticle significantly affected the ability of X. perforans in its production of xanthomonadin when compared with samples treated with micron-size copper or untreated. This study sheds new light on the potential utilization of this novel multi-site Cu/Zn hybrid nanoparticle for bacterial spot management.