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Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil.

ABSTRACT: Sinorhizobium meliloti CCNWSX0020, isolated from root nodules of Medicago lupulina growing in gold mine tailings in the northwest of China, displayed both copper resistance and growth promotion of leguminous plants in copper-contaminated soil. Nevertheless, the genetic and biochemical mechanisms responsible for copper resistance in S. meliloti CCNWSX0020 remained uncharacterized. To investigate genes involved in copper resistance, an S. meliloti CCNWSX0020 Tn5 insertion library of 14,000 mutants was created. Five copper-sensitive mutants, named SXa-1, SXa-2, SXc-1, SXc-2, and SXn, were isolated, and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Both SXa-1 and SXa-2 carried a transposon insertion in lpxXL (SM0020_18047), encoding the LpxXL C-28 acyltransferase; SXc-1 and SXc-2 carried a transposon insertion in merR (SM0020_29390), encoding the regulatory activator; SXn contained a transposon insertion in omp (SM0020_18792), encoding a hypothetical outer membrane protein. The results of reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that SM0020_05862, encoding an unusual P-type ATPase, was regulated by the MerR protein. Analysis of the genome sequence showed that this P-type ATPase did not contain an N-terminal metal-binding domain or a CPC motif but rather TPCP compared with CopA from Escherichia coli. Pot experiments were carried out to determine whether growth and copper accumulation of the host plant M. lupulina were affected in the presence of the wild type or the different mutants. Soil samples were subjected to three levels of copper contamination, namely, the uncontaminated control and 47.36 and 142.08 mg/kg, and three replicates were conducted for each treatment. The results showed that the wild-type S. meliloti CCNWSX0020 enabled the host plant to grow better and accumulate copper ions. The plant dry weight and copper content of M. lupulina inoculated with the 5 copper-sensitive mutants significantly decreased in the presence of CuSO4.

SUBMITTER: Li Z

PROVIDER: S-EPMC3957630 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil.

Li Zhefei Z Ma Zhanqiang Z Hao Xiuli X Rensing Christopher C Wei Gehong G

Applied and environmental microbiology 20140117 6

Sinorhizobium meliloti CCNWSX0020, isolated from root nodules of Medicago lupulina growing in gold mine tailings in the northwest of China, displayed both copper resistance and growth promotion of leguminous plants in copper-contaminated soil. Nevertheless, the genetic and biochemical mechanisms responsible for copper resistance in S. meliloti CCNWSX0020 remained uncharacterized. To investigate genes involved in copper resistance, an S. meliloti CCNWSX0020 Tn5 insertion library of 14,000 mutants ...[more]

PMID: 24441157

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Project description:The symbiosis of the highly metal-resistant Sinorhizobium meliloti CCNWSX0020 and Medicago lupulina has been considered an efficient tool for bioremediation of heavy metal-polluted soils. However, the metal resistance mechanisms of S. meliloti CCNWSX00200 have not been elucidated in detail. Here we employed a comparative transcriptome approach to analyze the defense mechanisms of S. meliloti CCNWSX00200 against Cu or Zn exposure. Six highly upregulated transcripts involved in Cu and Zn resistance were identified through deletion mutagenesis, including genes encoding a multicopper oxidase (CueO), an outer membrane protein (Omp), sulfite oxidoreductases (YedYZ), and three hypothetical proteins (a CusA-like protein, a FixH-like protein, and an unknown protein), and the corresponding mutant strains showed various degrees of sensitivity to multiple metals. The Cu-sensitive mutant (ΔcueO) and three mutants that were both Cu and Zn sensitive (ΔyedYZ, ΔcusA-like, and ΔfixH-like) were selected for further study of the effects of these metal resistance determinants on bioremediation. The results showed that inoculation with the ΔcueO mutant severely inhibited infection establishment and nodulation of M. lupulina under Cu stress, while inoculation with the ΔyedYZ and ΔfixH-like mutants decreased just the early infection frequency and nodulation under Cu and Zn stresses. In contrast, inoculation with the ΔcusA-like mutant almost led to loss of the symbiotic capacity of M. lupulina to even grow in uncontaminated soil. Moreover, the antioxidant enzyme activity and metal accumulation in roots of M. lupulina inoculated with all mutants were lower than those with the wild-type strain. These results suggest that heavy metal resistance determinants may promote bioremediation by directly or indirectly influencing formation of the rhizobium-legume symbiosis.IMPORTANCE Rhizobium-legume symbiosis has been promoted as an appropriate tool for bioremediation of heavy metal-contaminated soils. Considering the plant-growth-promoting traits and survival advantage of metal-resistant rhizobia in contaminated environments, more heavy metal-resistant rhizobia and genetically manipulated strains were investigated. In view of the genetic diversity of metal resistance determinants in rhizobia, their effects on phytoremediation by the rhizobium-legume symbiosis must be different and depend on their specific assigned functions. Our work provides a better understanding of the mechanism of heavy metal resistance determinants involved in the rhizobium-legume symbiosis, and in further studies, genetically modified rhizobia harboring effective heavy metal resistance determinants may be engineered for the practical application of rhizobium-legume symbiosis for bioremediation in metal-contaminated soils.

Project description:The double selection of environment adaptation and host specificity forced the diversification of rhizobia in nature. In the tropical region of China, Medicago polymorpha and Medicago lupulina are widely distributed, particularly in purple soil. However, the local distribution and diversity of rhizobia associated with these legumes has not been systematically investigated. To this end, root nodules of M. polymorpha and M. lupulina grown in purple soil at seven locations in Yunnan Province of China were collected for rhizobial isolation. The obtained rhizobia were characterized by RFLP of 16S-23S rRNA intergenic spacer, BOXAIR fingerprinting, and phylogeny of housekeeping and symbiosis genes. As result, a total of 91 rhizobial strains were classified into species Sinorhizobium medicae and S. meliloti, while three nodC gene types were identified among them. S. medicae containing nodC of type I was dominant in farmlands associated with M. polymorpha; while S. meliloti harboring nodC of type III was dominant in wild land nodulated by M. lupulina. For both rhizobial species, greater genetic diversity was detected in the populations isolated from their preferred host plant. A high level of genetic differentiation was observed between the two Sinorhizobium species, and gene flow was evident within the populations of the same species derived from different soil types, indicating that rhizobial evolution is likely associated with the soil features. To examine the effects of environmental features on rhizobial distribution, soil physicochemical traits and rhizobial genotypes were applied for constrained analysis of principle coordinates, which demonstrated that soil features like pH, nitrogen and sodium were the principle factors governing the rhizobial geographical distribution. Altogether, both S. medicae and S. meliloti strains could naturally nodulate with M. polymorpha and M. lupulina, but the rhizobium-legume symbiosis compatibility determined by both the host species and soil factors was also highlighted.

Project description:Myxococcus xanthus is a soil myxobacterium that exhibits a complex lifecycle with two multicellular stages: cooperative predation and development. During predation, myxobacterial cells produce a wide variety of secondary metabolites and hydrolytic enzymes to kill and consume the prey. It is known that eukaryotic predators, such as ameba and macrophages, introduce copper and other metals into the phagosomes to kill their prey by oxidative stress. However, the role of metals in bacterial predation has not yet been established. In this work, we have addressed the role of copper during predation of M. xanthus on Sinorhizobium meliloti. The use of biosensors, variable pressure scanning electron microscopy, high-resolution scanning transmission electron microscopy, and energy dispersive X ray analysis has revealed that copper accumulates in the region where predator and prey collide. This accumulation of metal up-regulates the expression of several mechanisms involved in copper detoxification in the predator (the P1 B-ATPase CopA, the multicopper oxidase CuoA and the tripartite pump Cus2), and the production by the prey of copper-inducible melanin, which is a polymer with the ability to protect cells from oxidative stress. We have identified two genes in S. meliloti (encoding a tyrosinase and a multicopper oxidase) that participate in the biosynthesis of melanin. Analysis of prey survivability in the co-culture of M. xanthus and a mutant of S. meliloti in which the two genes involved in melanin biosynthesis have been deleted has revealed that this mutant is more sensitive to predation than the wild-type strain. These results indicate that copper plays a role in bacterial predation and that melanin is used by the prey to defend itself from the predator. Taking into consideration that S. meliloti is a nitrogen-fixing bacterium in symbiosis with legumes that coexists in soils with M. xanthus and that copper is a common metal found in this habitat as a consequence of several human activities, these results provide clear evidence that the accumulation of this metal in the soil may influence the microbial ecosystems by affecting bacterial predatory activities.

Dataset Information

Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil.

Publications

Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil.

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