Rhizobium Inoculation Enhances the Resistance of Alfalfa and Microbial Characteristics in Copper-Contaminated Soil

Some studies have reported the importance of rhizobium in mitigating heavy metal toxicity, however, the regulatory mechanism of the alfalfa-rhizobium symbiosis to resist copper (Cu) stress in the plant-soil system through biochemical reactions is still unclear. This study assessed the effects of rhizobium (Sinorhizobium meliloti CCNWSX0020) inoculation on the growth of alfalfa and soil microbial characteristics under Cu-stress. Further, we determined the regulatory mechanism of rhizobium inoculation to alleviate Cu-stress in alfalfa through plant-soil system. The results showed that rhizobium inoculation markedly alleviated Cu-induced growth inhibition in alfalfa by increasing the chlorophyll content, height, and biomass, in addition to nitrogen and phosphorus contents. Furthermore, rhizobium application alleviated Cu-induced phytotoxicity by increasing the antioxidant enzyme activities and soluble protein content in tissues, and inhibiting the lipid peroxidation levels (i.e., malondialdehyde content). In addition, rhizobium inoculation improved soil nutrient cycling, which increased soil enzyme activities (i.e., β-glucosidase activity and alkaline phosphatase) and microbial biomass nitrogen. Both Pearson correlation coefficient analysis and partial least squares path modeling (PLS-PM) identified that the interactions between soil nutrient content, enzyme activity, microbial biomass, plant antioxidant enzymes, and oxidative damage could jointly regulate plant growth. This study provides comprehensive insights into the mechanism of action of the legume-rhizobium symbiotic system to mitigate Cu stress and provide an efficient strategy for phytoremediation of Cu-contaminated soils.

The copper resistome of group B Streptococcus reveals insight into the genetic basis of cellular survival during metal ion stress

In bacteria, copper (Cu) can support metabolic processes as an enzymatic cofactor but can also cause cell damage if present in excess, leading to intoxication. In group B Streptococcus (GBS) a system for control of Cu efflux based on the canonical cop operon supports survival during Cu stress. In some other bacteria, genetic systems additional to the cop operon are engaged during Cu stress and also contribute to Cu management. Here, we examined genetic systems beyond the cop operon in GBS for regions that contribute to survival of GBS in Cu stress using a forward genetic screen and probe of the entire bacterial genome. A high-density mutant library, generated using pGh9-ISS1, was used to expose GBS to Cu stress and compared to non-exposed controls en masse. Nine genes were identified as essential for GBS survival in Cu stress, whereas five genes constrained GBS growth in Cu stress. The genes encode varied factors including enzymes for metabolism, cell wall synthesis, transporters and global transcriptional regulators. Targeted mutation of the genes validated their roles in GBS resistance to Cu stress. Notably, several genes, including stp1, yceG, plyB and rfaB were also essential for resistance to Zn stress. Excepting copA, the genes identified are new to the area of bacterial metal ion intoxication. We conclude that a discrete and limited suite of genes beyond the cop operon in GBS contribute to a repertoire of mechanisms used to survive Cu stress in vitro and achieve cellular homeostasis.

Rhizobium inoculation enhanced the resistance of alfalfa and soil enzymatic activity in Cu-polluted soils

Although some studies have reported an important role of rhizobia in mitigating heavy metal toxicity, the regulatory mechanism of the alfalfa-rhizobium symbiosis system to resist copper (Cu) stress through biochemical reactions in the plant-soil system is still unclear. Hence, this study assessed the effects of rhizobium inoculation (i.e., Sinorhizobium meliloti CCNWSX0020) on the growth of alfalfa and soil enzyme activities under Cu stress. Our results showed that rhizobium inoculation markedly alleviated Cu-induced growth inhibition by increasing chlorophyll content, height and biomass and the contents of nitrogen and phosphorus in alfalfa. The content of malondialdehyde (MDA) was increased in both shoot and root of alfalfa under Cu stress. The application of rhizobium alleviated Cu-induced phytotoxicity by increasing the activity of antioxidant enzymes and soluble protein content of tissues and inhibiting the level of lipid peroxidation (i.e., MDA level). In addition, rhizobium inoculation improved soil nutrient cycling, increased soil enzyme activities (i.e., β-glucosidase activity and alkaline phosphatase) and microbial biomass nitrogen. Both Pearson correlation coefficient analysis and partial least squares path modeling (PLS-PM) identified that the interactions between soil nutrient content, enzyme activity, microbial biomass and plant antioxidant enzymes and oxidative damage could jointly regulate plant growth. This study provides comprehensive insights into the mechanism of action of the legume-rhizobium symbiosis system to mitigate Cu stress and provide an efficient strategy for phytoremediation of Cu-polluted soils.

Copper intoxication in group B Streptococcus triggers transcriptional activation of the cop operon that contributes to enhanced virulence during acute infection

Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY . Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments. Importance Understanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.

Proteome Changes Reveal the Protective Roles of Exogenous Citric Acid in Alleviating Cu Toxicity in Brassica napus L

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 μM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.

Environmental Conditions Modulate the Transcriptomic Response of Both Caulobacter crescentus Morphotypes to Cu Stress

Bacteria encounter elevated copper (Cu) concentrations in multiple environments, varying from mining wastes to antimicrobial applications of copper. As the role of the environment in the bacterial response to Cu ion exposure remains elusive, we used a tagRNA-seq approach to elucidate the disparate responses of two morphotypes of Caulobacter crescentus NA1000 to moderate Cu stress in a complex rich (PYE) medium and a defined poor (M2G) medium. The transcriptome was more responsive in M2G, where we observed an extensive oxidative stress response and reconfiguration of the proteome, as well as the induction of metal resistance clusters. In PYE, little evidence was found for an oxidative stress response, but several transport systems were differentially expressed, and an increased need for histidine was apparent. These results show that the Cu stress response is strongly dependent on the cellular environment. In addition, induction of the extracytoplasmic function sigma factor SigF and its regulon was shared by the Cu stress responses in both media, and its central role was confirmed by the phenotypic screening of a sigF::Tn5 mutant. In both media, stalked cells were more responsive to Cu stress than swarmer cells, and a stronger basal expression of several cell protection systems was noted, indicating that the swarmer cell is inherently more Cu resistant. Our approach also allowed for detecting several new transcription start sites, putatively indicating small regulatory RNAs, and additional levels of Cu-responsive regulation.

The physiological response of Ectomycorrhizal fungus Lepista sordida to Cd and Cu stress

Ectomycorrhizal fungi (ECMF) can develop the resistance of host plants to heavy metal stress. However, little is known about the response of ECMF to heavy metal exposure. In this study, the growth and physiological indices of Lepista sordida under Cd and Cu stress were studied. The growth of L. sordida on PDA medium under Cd and Cu stress was observed using scanning electron microscopy (SEM). After the addition of Cd and Cu to the medium, the mycelium started twisting, breaking, sticking together, and even dissolving. In the control group, a good and luxuriant mycelium growth of L. sordida along with the numerous clamp connections was observed. The mycelial biomass decreased with increasing concentrations of heavy metals in a liquid medium. The catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX) activities were also investigated, and the results showed that the Cd and Cu treatments caused a significant increase in the antioxidant enzyme activities. The contents of soluble protein, soluble sugar, and free proline in L. sordida were investigated, and it was found that the contents initially increased and then decreased with the increasing concentrations of Cd and Cu. However, the content of malondialdehyde (MDA) increased with the increasing concentrations of Cd and Cu. In conclusion, the present study provides a theoretical basis for the better utilization of Ectomycorrhizal fungal resources for the remediation of soil contaminated with heavy metal.