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Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China
JES-00142; No of Pages 9 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 4 ) XX X–XXX
Available online at www.sciencedirect.com
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Pin Xie1,2 , Xiuli Hao1 , Martin Herzberg2 , Yantao Luo1 , Dietrich H. Nies2 , Gehong Wei1,⁎
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Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China
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1. State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, China. E-mail: [email protected] 2. Molecular Microbiology, Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), 06120, Germany
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Article history:
To better understand the diversity of metal resistance genetic determinant from microbes that 17
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Received 2 April 2014
survived at metal tailings in northwest of China, a highly elevated level of heavy metal 18
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Revised 11 June 2014
containing region, genomic analyses was conducted using genome sequence of three 19
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Accepted 31 July 2014
native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium 20
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amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion 21
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Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel 22 Keywords:
as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is 23
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Plant growth promoting bacteria
expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA 24
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transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens 25
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Legume–rhizobia symbiosis
CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth 26
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promoting effects of these microbes on their hosts by nitrogen fixation and/or IAA secretion, 27 indicating a potential in-site phytoremediation usage in the mining tailing regions of China.
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Introduction
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Soil pollution is becoming one of the most severe environmental hazards in the past decades. Inorganic pollutants, in particular metals/metalloids, cannot be degraded biologically, and are ultimately indestructible and difficult to be removed from contaminated soils, threatening native microbial diversity and crop production. Surveys of plant species in long-term metalcontaminated environments showed legumes as the dominant portion in the various populations (Del Río et al., 2002; Hao et al., 2014). Legume plants, as an important component of agricultural ecosystems, play a crucial role in the food chain of animals and human consumption, maintaining soil fertility as well as the biogeochemical recycling of metal contaminated soil.
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For the healthy development of organisms, a suitable amount of transition metals must be acquired from the surrounding environments, especially in the metal concentrated soil, because essential minerals are necessary for various metabolic processes but toxic if in excess. Three different mechanisms might be used to adjust the cellular ion concentrations: (1) influx balance and efflux balance of transitional elements by ion transporters; (2) enzymatic detoxification by redox chemistry or covalent modification such as methylation; and (3) sequestration, binding of the toxic metal ion to proteins, polypeptides or other cellular components, whereas membrane transporters play a central role in maintaining metal ion resistance and homeostasis (Nies, 2007). A variety of gene families, which are likely to be involved
⁎ Corresponding author. E-mail: [email protected] (Gehong Wei).
http://dx.doi.org/10.1016/j.jes.2014.07.017 1001-0742/© 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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1. Materials and methods All sequence data and information mentioned in this study were acquired mainly from two sources: (1) relevant research papers on metal resistance and homeostasis related proteins in prokaryotes and eukaryotes, and (2) NCBI database. The gene/amino acid sequences of the three candidate strains have been downloaded in GenBank under the accession number AGVV00000000 for S. meliloti CCNWSX0020, AGSN0000000 for M. amorphae strain, and AGSM00000000 for A. tumefaciens CCNWGS0286, respectively (Li et al., 2012; Hao et al., 2012a,b). Phylogenetic analysis was conducted with the Neighbour Joining Method as implemented in the Molecular Evolutionary Genetic Analysis package (MEGA5) (Tamura et al., 2011). The reliability of the branches was estimated by bootstrap analysis with 1000 bootstrap replications. Phylogenetic tree was constructed for CDF (Cation Diffusion Facilitator) family using the key putative or characterized proteins from the three strains and other organisms (Table S1). Multiple alignments of putative “CopA” were done with ClustalW to identify the conserved domain of each protein. The logo sequences were generated using texshade software (Eric, 2000). Transmembrane segments of zinc transporters were identified using SMART and SOSUI signal (Schultz et al., 1998). The accession numbers of amino acid sequences used in study were listed in the Supplementary file.
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2. Metal resistance and homeostasis comparison of the three metal tolerant bacteria
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2.1. Copper
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Copper is an essential redoxactive transitional element that plays a significant role in many physiological processes, but is
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toxic at high concentration (Rapisarda et al., 2002; Letelier et al., 2010). Organisms have evolved several strategies to regulate copper homeostasis on the level of uptake, intracellular distribution and efflux. Copper-transporting P-type ATPase has been identified as one of the best understood copper homeostasis factors in organisms, and displays a high degree of conservation of copper trafficking system from bacteria to humans (Inmaculada, 2005; Pilon, 2011). According to the genome sequence of the reported strains, putative copA or copA-like genes are expected to be responsible for copper tolerance (SM0020_11415, SM0020_05912, SM0020_05727 in S. meliloti, MEA186_07644, MEA186_35439 in M. amorphae, but no putative gene in A. tumefaciens). Regarding the genomic environment of putative copA genes in the two strains, putative copG gene, previously being reported to be involved in the synthesis of prosthetic copper clusters in enzymes and deliver superfluous Cu(I) to the CopA exporter, has been numbered in these two strains as SM0020_30972 and MEA186_32635, respectively (Rensing et al., 2000). Moreover, cueR, a Cu(I)-responsive MerR-like transcriptional regulator (SM0020_11410, MEA186_07594) has also been identified in the two strains, but with two additional copies of periplasmic blue copper oxidase precursor genes (SM0020_18797, SM0020_23122) in S. meliloti, named as cueO. This resembled CopA/CueO system in S. meliloti is presumed to pump out excess Cu(I) from the cytoplasm into the periplasm via CopA transporters, accompanying with CueO oxidizing Cu(I) to Cu(II) in the periplasm thereby reducing copper toxicity similar to that in Escherichia coli (Rensing et al., 2000; Dmitriev et al., 2006; Li et al., 2014). Additionally, several copper chaperones, copper-binding proteins, unspecific divalent cation transporters, and proteins involved in the cytochrome oxidase of the respiratory chain may participate in copper transport and sequestration, similar to the situation in A. tumefaciens. The deduced protein sequences of the respective CopA-like transporters (EHK77876, EHK78957 and EHK78920 in S. meliloti, EHH12654 and EHH02252 in M. amorphae), were compared with CopA sequence of E. coli (EDV65660) using BLAST (Fig. 1). Sequence features of these five putative copper transporters clearly demonstrated the characteristic P-type domain arrangement in the order of the aspartyl kinase domain (A-domain), phosphorylation (P-domain) and the nucleotide binding domain (N-domain), according to the conserved motifs for each domain (S/TGES, DKTG, GDGxNDxP for A, P, N, respectively) (Fig. 1) (Argüello et al., 2007). Similar to CopA from E. coli, EHK77876 has one copy of a “GMxCxxC” motif, which represents a metal binding domain (MBD) in the N-terminus, illustrating a specific copper transporter (Fig. 1) (Gupta and Lutsenko, 2012). However, the other four proteins only contain “CP” motif in the N-terminal for metal-binding, which still needs further investigation. Briefly, the management of copper resistance in M. amorphae and S. meliloti is achieved by the CopA-like P-type ATPase efflux process, assisted by a complex homeostasis regulation network.
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in ion transport in prokaryotes and eukaryotes, have already been identified by genetic and molecular techniques (Nies, 2003). However, the mechanism used and particularly the co-ordination of regulatory responses of metal resistance and homeostasis in organisms vary significantly between species. During the last few years the genomes of a large number of rhizobia (a member of PGPB) from different species have been sequenced. The availability of genomes for two metal tolerant rhizobia, Sinorhizobium meliloti CCNWSX0020 (from Medicago lupulina), Mesorhizobium amorphae CCNWGS0123 (from R. pseudoacacia) and Agrobacterium tumefaciens CCNWGS0286 (a PGPB from Robinia pseudoacacia) and in our workgroup, provides the unique possibility for species comparison of metal resistance determinants among species (Li et al., 2012; Hao et al., 2012a,b). This review highlights the general mechanisms of the three metal tolerant bacteria against copper, zinc, nickel as well as chromium based on the genomic analysis. This work is necessary for a better understanding of the genetic diversity of metal resistance determinant among rhizobial strains from metal tailings in the northwest of China, which is such a highly polluted area that limited number of plants and microbial species could survive (including rhizobia–legume symbiosis). This would be very helpful to further explore how the metal resistant determinant from the bacterial side influences the legume plant–rhizobia interaction process.
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2.2. Zinc
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Zinc plays an amazing number of critical roles in all organisms, 179 a good example of the diverse biological utility of metal ion 180 (Rhodes and Klug, 1993). Similar to the situation with copper, 181 Q26
Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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Fig. 1 – Conserved domains in the predicted proteins copper transporting P-type ATPases in S. meliloti, M. amorphae, and CopA in E. coli. P-, A-, N-domain are shorted for the phosphorylation domain, the aspartyl kinase domain and the nucleotide binding domain, respectively.
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shares 45% amino acid identity with ZitB of E. coli (EKK89753), putatively mediating efflux of zinc across the plasma membrane (Chao and Fu, 2004), whereas EHH03182 (from A. tumefaciens) and EHK76549 (from S. meliloti) were clustered into the CzcD-like protein. Taking ZitB in M. amorphae for example, it displays a common structural characteristics, with six TMS and N- and C-terminal H/S-rich motifs (predicted to extend into the cytoplasm), as well as a CDF signature sequence between TM I and TM II (Fig. 3). The conserved motifs “HMLXXD” and “HVLGD” at the TMII and TMV are an evidence for a ZitB-like protein in the Zn-CDF clusters (Barbara et al., 2007). Eukaryotic members of the Zn-CDF s (e.g., Zrc-like, Msc-like), in contrast, show a His-rich region between TM IV and TM V, which is also predicted to be within the cytoplasm (Paulsen and Saie, 1997). Zn-CDF members may differ in the range of their substrate specificity. Known members of ZitB-like, Msc-like, Zrg17-like, and Znt-like clusters are highly specific for zinc translocation while DME-like and Zrc-like proteins are also involved in cobalt and cadmium trafficking (Barbara et al., 2007). As an ancient protein family, protein members of CDF family evolved with environment substrate specific selection and adaptation, but not related to the organism taxonomy.
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zinc can be toxic if in excess (Wu et al., 1992). Generally, the zinc resistance and detoxification in bacteria are achieved via three efflux mechanisms: P-type efflux ATPases, CDF proteins and RND-driven (resistance nodulation division family) transporters (Nies, 1999). Genes putatively involved in zinc homeostasis were identified on the genome of the three PGPBs, including one or more putative heavy metal transporting ATPases (SM0020_05862, SM0020_22747 in S. meliloti, MEA186_07749, MEA186_07989 in M. amorphae, and ATCR1_00750, ATCR1_21067, ATCR1_21784, ATCR1_02265), which was proved to be regulated by MerR-type regulators named as ZntR1 in A. tumefaciens (Hao et al., 2012b), one gene encoding for ion transporter from CDF (cation–diffusion facilitator) family (SM0020_17742 in S. meliloti, MEA186_00250 in M. amorphae, and ATCR1_22014 in A. tumefaciens), but no metal transporters from the RND family in these three isolates. Additionally, zinc ABC transporters (ZnuABC) and ZntA transporters have also been identified in A. tumefaciens, and numbered as ATCR1_02230, ATCR1_02220, ATCR1_02225, ATCR1_24860, ATCR1_24865, and ATCR1_24870 in order. Putative zinc CDF transporters from the three strains were classified in Fig. 2. Among them, EHH14095 from M. amorphae
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Fig. 2 – Phylogenetic analysis of CDF family proteins from M. amorphae, S. meliloti and A. tumefaciens and (accession numbers EHH14095, EHK76549 and EHH03182 in order). Phylogenetic tree was constructed using the neighbor joining method with protein sequences of CDF family from NCBI database, and accession numbers were available in Table S1. Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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Fig. 3 – TMS distribution and conserved domains of putative ZitB in M. amorphae, and E. coli (accession numbers EHH14095 and ACC73839 in order).
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Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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As one of the most broad range used of element, biological beneficial functions of chromium are only known for Cr(III) in higher organisms while there is no known beneficial influence of chromate in the whole biosphere (Park et al., 2006). Bacteria have developed several strategies against chromate stress, mostly based on chromate efflux system and reduction reaction of Cr(VI) to Cr(III) (Ramírez-Díaz et al., 2001; Cervantes et al., 1990; Nies et al., 1990). Due to the genomic prediction of the three strains, putative chrA genes are expected to be responsible for chromium tolerance (SM0020_07772 in S. meliloti, MEA186_33314 in M. amorphae, ATCR1_19096 and ATCR1_03154 in A. tumefaciens). Additionally, MEA186_33319 from M. amorphae, encoding for ChrB, acts as regulators of ChrA-mediated chromate resistance similar to that in Cupriavidus metallidurans (Juhnke et al., 2002). Further analysis of amino acid alignment revealed that the ChrA protein (EHH57475, 461 amino acids, 10 TMS) from M. amorphae strain shows 54% similarity to ChrA (EHK78617) from S. meliloti, 25% to C. metallidurans (CAI30234), and 26% to
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3. Metal resistance determinants in plant growth promoting bacteria are necessary for the development and growth of legumes
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Nickel, as the 24th most abundant element in the earth's crust, consequently, is necessary for a variety of metabolic processes (Mulrooney and Hausinger, 2003). However, organisms have developed a number of devices to balance nickel level in the cellular level, because high concentrations of Ni(II) can be toxic to cells (Gajewska et al., 2006). Based on the genome sequence of M. amorphae, nickel is imported presumably by the high affinity nickel transporter from NiCoT family (like HoxN, NixA, and HupN), encoded by MEA186_10020. Even though without the nickel efflux systems (e.g., CnrCBA (cobalt–nickel resistance), NccCBA (nickel–cobalt–cadmium) and NreB (nickel resistance)) in M. amorphae, Ni(II) in the cell could be bound by nickel chaperone and delivered subsequently to its target protein. Two copies of hupE genes (MEA186_23401 and MEA186_06578), were expected to be involved in nickel sequestration in M. amorphae, due to the fact that HupE has been verified as such a nickel chaperone in the (hupSLCDEFGHIJK) cluster in Rhizobium leguminosarum (Brito et al., 2010). Meanwhile, hypB gene, numbered as MEA186_29702, is a hydrogenase accessory protein that is involved in nickel insertion into maturating hydrogenase in other rhizobia (Olson and Maier, 2000). Nickel sequestering at the N-terminal histidine rich region of HypB could also be considered as a potential supplemental pathway in the nickel homeostasis regulation in this soil bacterium. However, situations were totally different in other two bacteria. With a genomic insight, cbtA (previously considered as a secondary nickel transporter), numbered as ATCR1_22049, is expected to be involved in nickel transport in A. tumefaciens, assisted by a MerR-like transcriptional regulator (ATCR1_00740) (Eitinger et al., 2005). While no nickel homeostasis related genes were predicted in the genome of S. meliloti.
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PGPB assist the growth of plants in metal enriched soil ecosystem, due to the production of phytohormone (e.g., indole-3-acetic acid, IAA), ACC-deaminase and siderophores, phosphorus solubilization, and the interesting nitrogen-fixing process (Han et al., 2005). These properties imply the potential usage of PGPB in the phytoremediation of metal/metalloid contaminated soil (Denton, 2007). Rhizobia, as a subset of PGPB, thus play a key role in the phytoremediation by legume plants (Safronova et al., 2011; Hao et al., 2014). Indeed, many rhizobial strains have been reported to protect their various host legumes from metals toxicity, pathogens and improve plant growth under stressed conditions (Gupta et al., 2004; Wani et al., 2008a, b,c; Jian et al., 2009), but few of them has been used as a material for the metal resistant determinant investigation. There's still no sufficient molecular evidence to explain how the microbe– legume symbiosis survives at metal stressed soil. Beginning with the metal resistant endophytic bacteria of legume nodules, it's necessary to investigate the effect of heavy metal resistance determinants from the three PGPBs exerting on the growth and the development of legume plants, and to explore the potential application of this microbe–legume symbiosis to heavy metal enriched soil in-site remediation and soil fertility restoration in the mining tailing in northwest of China. In our greenhouse study, the metal resistance determinant in rhizobia was proved to be required for the functional symbiosis formation in the stress of heavy metals. In M. amorphae, chrA has been founded as a necessity for the formation of active nodules in the root of R. pseudoacacia at 25 mg/kg chromate stress, maintaining a regular expression level of leghemoglobin in the nodules and attaining a relative higher nitrogen accumulation than that of the △ chrA inoculation and un-inoculated control (unpublished). Similar results have also been observed in the nodules of inoculated Lentil, which attains a higher shoot N content and leghemoglobin content than the un-inoculated plants, but no results for the metal sensitive inoculated treatment (Wani et al., 2008c). Regarding M. lupulina, with similar nodulation frequency, there's no significant difference on the dry weight inoculated either with the S. meliloti parent or mutant strain (Li et al., 2014). Interestingly, S. meliloti could produce IAA under copper stressed conditions, with lower IAA production level achieved by copper sensitive mutants than that of the wild type strain (Li et al., 2014). Similarly, studies on the IAA production by A. tumefaciens found that IAA amount decreased with the increasing concentration of
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P. aeruginosa (AEQ93501). The broadly distributed and highly conserved motifs “FGGP” and “PGP” of these predicted ChrA proteins suggest a closely evolutionary relationship with other bacteria, indicating an ancient origin of the CHR family. Chromate may also be reduced in higher plants to Cr(III) by Cr reductases, even though no discrete enzymes have been identified (Lytle et al., 1998). There is, however, no such gene encoding a possible chromate reductase in the three PGRB strains. Thus, these three isolates may detoxify chromate mainly via the ATP-catalyzed ChrA efflux process.
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To sum up, zinc resistance in the three strains was realized via the cooperation the CDF family transporter and unspecific P-type ATP-catalyzed efflux process.
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Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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copper and zinc in the medium. R. pseudoacacia growth was much more greatly enhanced by inoculation of IAA production 342 strain A. tumefaciens compared to that of zinc sensitive mutant 343 (Hao et al., 2012a). Based on these findings, it can only be 344 concluded that the metal resistant genes may interfere with the 345 production of phytohorme of these two PGPBs via metal 346 homeostasis regulation. However, it's still needed to elucidate 347 how these metal resistance determinants interfere with the 348 nodulation and nitrogen fixation, the IAA synthesis, even some 349 un-covered but ion-mediated metabolic pathway. 350 Considering the advantages of using legumes as a resource 351 for phytoremediation (or phytostabilization), metal accumu352 lation capability of the legume plants inoculated by metal 353 resistant PGPB were analyzed. Actually, as the increasing of 354 metal concentrations in soil, the ion content in the tissues of 355 legume plants. While, R. pseudoacacia plants infected with 356 M. amorphae CCNWGS0123 led to a higher increment of 357 chromium content in the roots, but infection with the ΔchrA 358 mutant did not (unpublished). Regarding M. lupulina, plants 359 inoculated with copper-sensitive mutant accumulated much 360 less the metal in the tissues, compared to the plants inoculated 361 Q60 with S. meliloti CCNWSX0020 (Li et al., 2014). Similarly, Lupinus 362 albus inoculated with Bradyrhizobium L-7AH (metal resistant 363 strain) was able to accumulate large amounts of Hg in their 364 roots (Quiñones et al., 2013). Under metal stressed conditions, 365 this phenomenon that was explained by a greater biomass of 366 legumes was acquired via the inoculation with metal resistant 367 bacteria, but metal concentrations were redistributed among 368 plant tissues (Hao et al., 2014). It exactly indicates that 369 this legume–microbe system is suitable for the usage of 370 phytoremediation of metal contaminated soil, especially in 371 mine tailing regions.
contents (essential metals such as Zn, Cu, Fe, Mn, Co, as well as Ni; toxic heavy metals such as Cd and Cr) and metal toxicity of legumes, compared to that in other grains like rice, wheat, and maize. With a potential metal resistance and homeostasis network of the three metal tolerant PGPBs, the interaction of these microbes and their host plant symbiosis with respect to trace element homeostasis could be explored in several fields. Going in a top-down approach from an agricultural ecosystem to the sub-cellular level, it's necessary: (1) to understand the metal composition of the respective soil and how this influences the metal–microbe–plant interaction; (2) to metal resistance and homeostasis in the plant and the interacting bacterium; and (3) to how the various metal resistance determinants influence the metal distribution and interplay of in the plant and bacterial cell. This is especially true for soils that are in the mine tailing and contaminated with transition metals (Chen et al., 2012; Hanikenne and Nouet, 2011). Certainly, to better understand the complicated metal homeostasis of the microbe–plant symbiosis, from trace metal uptake to transport, from accumulation to detoxification, and from plant growth promotion to inhibition of rhizobium, further studies are needed both at biochemical and molecular levels. Molecular-genetic and physic-chem techniques will be applied to deeply study the interplay of the three PGRBs and their hosts.
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4. Discussion
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In the view of the whole genomic picture of the three metal resistant PGPB, M. amorphae contains protein members from P-type ATPase, CDF, HupE/UreJ and CHR family, which are expected to be involved in copper, zinc, nickel as well as chromate resistance and homeostasis. While, S. meliloti and A. tumefaciens exhibit a much more competent trafficking and detoxification network for copper and zinc homeostasis, respectively. Simon and Phung (1996) assumed that microbial metal resistance systems arose shortly after prokaryote life started. In fact, the resistance systems have been found in essentially all bacterial types, even though not precisely the same due to the substrate specific selection and environment adaptation (Nies, 2003). Lakzian et al. (2007) demonstrated that elevated levels of heavy metals induced the natural plasmid transferring between rhizobia. This study plays a crucial role in conferring enhanced and diversified metal tolerance of these dominant microbes in the mine tailings in Northwest of China. Legumes are such an important candidate that is 3rd largest family of flowering plants, widely existing in the forms of vegetable, oilseed, medicinal crops, animal feed (Ahmad et al., 2012). As a complement of cereals, it enters the human food chain and agriculture system directly or indirectly. However, little work has been performed on the determination of metal
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This work systematically presented the putative metal resistance determinant in the three PGPBs, containing transporter members from P-type ATPase, CDF, HupE/UreJ and CHR family involved in copper, zinc, nickel as well as chromate resistance. It was a very competent and intricate multi-metal homeostasis network, ensuring the survival capability of these isolates in the heavy polluted ecosystems. Together with their hosts, the M. amophae/A. tumefaciens–R. pseudoacacia, M. lupulina–S. meliloti symbiosis provides a potential tool for metal removal and soil fertility restoration in the metal tailing area. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jes.2014.07.017.
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This work was supported by the National Science Foundation of China (Nos. 31125007, 31370142), the National High Technology Research and Development Program (863) of China (No. 2012AA101402).
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Pin Xie1,2 , Xiuli Hao1 , Martin Herzberg2 , Yantao Luo1 , Dietrich H. Nies2 , Gehong Wei1,⁎
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Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China
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1. State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, China. E-mail: [email protected] 2. Molecular Microbiology, Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), 06120, Germany
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Article history:
To better understand the diversity of metal resistance genetic determinant from microbes that 17
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Received 2 April 2014
survived at metal tailings in northwest of China, a highly elevated level of heavy metal 18
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Revised 11 June 2014
containing region, genomic analyses was conducted using genome sequence of three 19
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native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium 20
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amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion 21
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Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel 22 Keywords:
as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is 23
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expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA 24
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transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens 25
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Legume–rhizobia symbiosis
CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth 26
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promoting effects of these microbes on their hosts by nitrogen fixation and/or IAA secretion, 27 indicating a potential in-site phytoremediation usage in the mining tailing regions of China.
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Introduction
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Soil pollution is becoming one of the most severe environmental hazards in the past decades. Inorganic pollutants, in particular metals/metalloids, cannot be degraded biologically, and are ultimately indestructible and difficult to be removed from contaminated soils, threatening native microbial diversity and crop production. Surveys of plant species in long-term metalcontaminated environments showed legumes as the dominant portion in the various populations (Del Río et al., 2002; Hao et al., 2014). Legume plants, as an important component of agricultural ecosystems, play a crucial role in the food chain of animals and human consumption, maintaining soil fertility as well as the biogeochemical recycling of metal contaminated soil.
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For the healthy development of organisms, a suitable amount of transition metals must be acquired from the surrounding environments, especially in the metal concentrated soil, because essential minerals are necessary for various metabolic processes but toxic if in excess. Three different mechanisms might be used to adjust the cellular ion concentrations: (1) influx balance and efflux balance of transitional elements by ion transporters; (2) enzymatic detoxification by redox chemistry or covalent modification such as methylation; and (3) sequestration, binding of the toxic metal ion to proteins, polypeptides or other cellular components, whereas membrane transporters play a central role in maintaining metal ion resistance and homeostasis (Nies, 2007). A variety of gene families, which are likely to be involved
⁎ Corresponding author. E-mail: [email protected] (Gehong Wei).
http://dx.doi.org/10.1016/j.jes.2014.07.017 1001-0742/© 2014 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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1. Materials and methods All sequence data and information mentioned in this study were acquired mainly from two sources: (1) relevant research papers on metal resistance and homeostasis related proteins in prokaryotes and eukaryotes, and (2) NCBI database. The gene/amino acid sequences of the three candidate strains have been downloaded in GenBank under the accession number AGVV00000000 for S. meliloti CCNWSX0020, AGSN0000000 for M. amorphae strain, and AGSM00000000 for A. tumefaciens CCNWGS0286, respectively (Li et al., 2012; Hao et al., 2012a,b). Phylogenetic analysis was conducted with the Neighbour Joining Method as implemented in the Molecular Evolutionary Genetic Analysis package (MEGA5) (Tamura et al., 2011). The reliability of the branches was estimated by bootstrap analysis with 1000 bootstrap replications. Phylogenetic tree was constructed for CDF (Cation Diffusion Facilitator) family using the key putative or characterized proteins from the three strains and other organisms (Table S1). Multiple alignments of putative “CopA” were done with ClustalW to identify the conserved domain of each protein. The logo sequences were generated using texshade software (Eric, 2000). Transmembrane segments of zinc transporters were identified using SMART and SOSUI signal (Schultz et al., 1998). The accession numbers of amino acid sequences used in study were listed in the Supplementary file.
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2. Metal resistance and homeostasis comparison of the three metal tolerant bacteria
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2.1. Copper
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Copper is an essential redoxactive transitional element that plays a significant role in many physiological processes, but is
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toxic at high concentration (Rapisarda et al., 2002; Letelier et al., 2010). Organisms have evolved several strategies to regulate copper homeostasis on the level of uptake, intracellular distribution and efflux. Copper-transporting P-type ATPase has been identified as one of the best understood copper homeostasis factors in organisms, and displays a high degree of conservation of copper trafficking system from bacteria to humans (Inmaculada, 2005; Pilon, 2011). According to the genome sequence of the reported strains, putative copA or copA-like genes are expected to be responsible for copper tolerance (SM0020_11415, SM0020_05912, SM0020_05727 in S. meliloti, MEA186_07644, MEA186_35439 in M. amorphae, but no putative gene in A. tumefaciens). Regarding the genomic environment of putative copA genes in the two strains, putative copG gene, previously being reported to be involved in the synthesis of prosthetic copper clusters in enzymes and deliver superfluous Cu(I) to the CopA exporter, has been numbered in these two strains as SM0020_30972 and MEA186_32635, respectively (Rensing et al., 2000). Moreover, cueR, a Cu(I)-responsive MerR-like transcriptional regulator (SM0020_11410, MEA186_07594) has also been identified in the two strains, but with two additional copies of periplasmic blue copper oxidase precursor genes (SM0020_18797, SM0020_23122) in S. meliloti, named as cueO. This resembled CopA/CueO system in S. meliloti is presumed to pump out excess Cu(I) from the cytoplasm into the periplasm via CopA transporters, accompanying with CueO oxidizing Cu(I) to Cu(II) in the periplasm thereby reducing copper toxicity similar to that in Escherichia coli (Rensing et al., 2000; Dmitriev et al., 2006; Li et al., 2014). Additionally, several copper chaperones, copper-binding proteins, unspecific divalent cation transporters, and proteins involved in the cytochrome oxidase of the respiratory chain may participate in copper transport and sequestration, similar to the situation in A. tumefaciens. The deduced protein sequences of the respective CopA-like transporters (EHK77876, EHK78957 and EHK78920 in S. meliloti, EHH12654 and EHH02252 in M. amorphae), were compared with CopA sequence of E. coli (EDV65660) using BLAST (Fig. 1). Sequence features of these five putative copper transporters clearly demonstrated the characteristic P-type domain arrangement in the order of the aspartyl kinase domain (A-domain), phosphorylation (P-domain) and the nucleotide binding domain (N-domain), according to the conserved motifs for each domain (S/TGES, DKTG, GDGxNDxP for A, P, N, respectively) (Fig. 1) (Argüello et al., 2007). Similar to CopA from E. coli, EHK77876 has one copy of a “GMxCxxC” motif, which represents a metal binding domain (MBD) in the N-terminus, illustrating a specific copper transporter (Fig. 1) (Gupta and Lutsenko, 2012). However, the other four proteins only contain “CP” motif in the N-terminal for metal-binding, which still needs further investigation. Briefly, the management of copper resistance in M. amorphae and S. meliloti is achieved by the CopA-like P-type ATPase efflux process, assisted by a complex homeostasis regulation network.
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in ion transport in prokaryotes and eukaryotes, have already been identified by genetic and molecular techniques (Nies, 2003). However, the mechanism used and particularly the co-ordination of regulatory responses of metal resistance and homeostasis in organisms vary significantly between species. During the last few years the genomes of a large number of rhizobia (a member of PGPB) from different species have been sequenced. The availability of genomes for two metal tolerant rhizobia, Sinorhizobium meliloti CCNWSX0020 (from Medicago lupulina), Mesorhizobium amorphae CCNWGS0123 (from R. pseudoacacia) and Agrobacterium tumefaciens CCNWGS0286 (a PGPB from Robinia pseudoacacia) and in our workgroup, provides the unique possibility for species comparison of metal resistance determinants among species (Li et al., 2012; Hao et al., 2012a,b). This review highlights the general mechanisms of the three metal tolerant bacteria against copper, zinc, nickel as well as chromium based on the genomic analysis. This work is necessary for a better understanding of the genetic diversity of metal resistance determinant among rhizobial strains from metal tailings in the northwest of China, which is such a highly polluted area that limited number of plants and microbial species could survive (including rhizobia–legume symbiosis). This would be very helpful to further explore how the metal resistant determinant from the bacterial side influences the legume plant–rhizobia interaction process.
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2.2. Zinc
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Zinc plays an amazing number of critical roles in all organisms, 179 a good example of the diverse biological utility of metal ion 180 (Rhodes and Klug, 1993). Similar to the situation with copper, 181 Q26
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Fig. 1 – Conserved domains in the predicted proteins copper transporting P-type ATPases in S. meliloti, M. amorphae, and CopA in E. coli. P-, A-, N-domain are shorted for the phosphorylation domain, the aspartyl kinase domain and the nucleotide binding domain, respectively.
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shares 45% amino acid identity with ZitB of E. coli (EKK89753), putatively mediating efflux of zinc across the plasma membrane (Chao and Fu, 2004), whereas EHH03182 (from A. tumefaciens) and EHK76549 (from S. meliloti) were clustered into the CzcD-like protein. Taking ZitB in M. amorphae for example, it displays a common structural characteristics, with six TMS and N- and C-terminal H/S-rich motifs (predicted to extend into the cytoplasm), as well as a CDF signature sequence between TM I and TM II (Fig. 3). The conserved motifs “HMLXXD” and “HVLGD” at the TMII and TMV are an evidence for a ZitB-like protein in the Zn-CDF clusters (Barbara et al., 2007). Eukaryotic members of the Zn-CDF s (e.g., Zrc-like, Msc-like), in contrast, show a His-rich region between TM IV and TM V, which is also predicted to be within the cytoplasm (Paulsen and Saie, 1997). Zn-CDF members may differ in the range of their substrate specificity. Known members of ZitB-like, Msc-like, Zrg17-like, and Znt-like clusters are highly specific for zinc translocation while DME-like and Zrc-like proteins are also involved in cobalt and cadmium trafficking (Barbara et al., 2007). As an ancient protein family, protein members of CDF family evolved with environment substrate specific selection and adaptation, but not related to the organism taxonomy.
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zinc can be toxic if in excess (Wu et al., 1992). Generally, the zinc resistance and detoxification in bacteria are achieved via three efflux mechanisms: P-type efflux ATPases, CDF proteins and RND-driven (resistance nodulation division family) transporters (Nies, 1999). Genes putatively involved in zinc homeostasis were identified on the genome of the three PGPBs, including one or more putative heavy metal transporting ATPases (SM0020_05862, SM0020_22747 in S. meliloti, MEA186_07749, MEA186_07989 in M. amorphae, and ATCR1_00750, ATCR1_21067, ATCR1_21784, ATCR1_02265), which was proved to be regulated by MerR-type regulators named as ZntR1 in A. tumefaciens (Hao et al., 2012b), one gene encoding for ion transporter from CDF (cation–diffusion facilitator) family (SM0020_17742 in S. meliloti, MEA186_00250 in M. amorphae, and ATCR1_22014 in A. tumefaciens), but no metal transporters from the RND family in these three isolates. Additionally, zinc ABC transporters (ZnuABC) and ZntA transporters have also been identified in A. tumefaciens, and numbered as ATCR1_02230, ATCR1_02220, ATCR1_02225, ATCR1_24860, ATCR1_24865, and ATCR1_24870 in order. Putative zinc CDF transporters from the three strains were classified in Fig. 2. Among them, EHH14095 from M. amorphae
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Fig. 2 – Phylogenetic analysis of CDF family proteins from M. amorphae, S. meliloti and A. tumefaciens and (accession numbers EHH14095, EHK76549 and EHH03182 in order). Phylogenetic tree was constructed using the neighbor joining method with protein sequences of CDF family from NCBI database, and accession numbers were available in Table S1. Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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Fig. 3 – TMS distribution and conserved domains of putative ZitB in M. amorphae, and E. coli (accession numbers EHH14095 and ACC73839 in order).
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Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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As one of the most broad range used of element, biological beneficial functions of chromium are only known for Cr(III) in higher organisms while there is no known beneficial influence of chromate in the whole biosphere (Park et al., 2006). Bacteria have developed several strategies against chromate stress, mostly based on chromate efflux system and reduction reaction of Cr(VI) to Cr(III) (Ramírez-Díaz et al., 2001; Cervantes et al., 1990; Nies et al., 1990). Due to the genomic prediction of the three strains, putative chrA genes are expected to be responsible for chromium tolerance (SM0020_07772 in S. meliloti, MEA186_33314 in M. amorphae, ATCR1_19096 and ATCR1_03154 in A. tumefaciens). Additionally, MEA186_33319 from M. amorphae, encoding for ChrB, acts as regulators of ChrA-mediated chromate resistance similar to that in Cupriavidus metallidurans (Juhnke et al., 2002). Further analysis of amino acid alignment revealed that the ChrA protein (EHH57475, 461 amino acids, 10 TMS) from M. amorphae strain shows 54% similarity to ChrA (EHK78617) from S. meliloti, 25% to C. metallidurans (CAI30234), and 26% to
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Nickel, as the 24th most abundant element in the earth's crust, consequently, is necessary for a variety of metabolic processes (Mulrooney and Hausinger, 2003). However, organisms have developed a number of devices to balance nickel level in the cellular level, because high concentrations of Ni(II) can be toxic to cells (Gajewska et al., 2006). Based on the genome sequence of M. amorphae, nickel is imported presumably by the high affinity nickel transporter from NiCoT family (like HoxN, NixA, and HupN), encoded by MEA186_10020. Even though without the nickel efflux systems (e.g., CnrCBA (cobalt–nickel resistance), NccCBA (nickel–cobalt–cadmium) and NreB (nickel resistance)) in M. amorphae, Ni(II) in the cell could be bound by nickel chaperone and delivered subsequently to its target protein. Two copies of hupE genes (MEA186_23401 and MEA186_06578), were expected to be involved in nickel sequestration in M. amorphae, due to the fact that HupE has been verified as such a nickel chaperone in the (hupSLCDEFGHIJK) cluster in Rhizobium leguminosarum (Brito et al., 2010). Meanwhile, hypB gene, numbered as MEA186_29702, is a hydrogenase accessory protein that is involved in nickel insertion into maturating hydrogenase in other rhizobia (Olson and Maier, 2000). Nickel sequestering at the N-terminal histidine rich region of HypB could also be considered as a potential supplemental pathway in the nickel homeostasis regulation in this soil bacterium. However, situations were totally different in other two bacteria. With a genomic insight, cbtA (previously considered as a secondary nickel transporter), numbered as ATCR1_22049, is expected to be involved in nickel transport in A. tumefaciens, assisted by a MerR-like transcriptional regulator (ATCR1_00740) (Eitinger et al., 2005). While no nickel homeostasis related genes were predicted in the genome of S. meliloti.
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PGPB assist the growth of plants in metal enriched soil ecosystem, due to the production of phytohormone (e.g., indole-3-acetic acid, IAA), ACC-deaminase and siderophores, phosphorus solubilization, and the interesting nitrogen-fixing process (Han et al., 2005). These properties imply the potential usage of PGPB in the phytoremediation of metal/metalloid contaminated soil (Denton, 2007). Rhizobia, as a subset of PGPB, thus play a key role in the phytoremediation by legume plants (Safronova et al., 2011; Hao et al., 2014). Indeed, many rhizobial strains have been reported to protect their various host legumes from metals toxicity, pathogens and improve plant growth under stressed conditions (Gupta et al., 2004; Wani et al., 2008a, b,c; Jian et al., 2009), but few of them has been used as a material for the metal resistant determinant investigation. There's still no sufficient molecular evidence to explain how the microbe– legume symbiosis survives at metal stressed soil. Beginning with the metal resistant endophytic bacteria of legume nodules, it's necessary to investigate the effect of heavy metal resistance determinants from the three PGPBs exerting on the growth and the development of legume plants, and to explore the potential application of this microbe–legume symbiosis to heavy metal enriched soil in-site remediation and soil fertility restoration in the mining tailing in northwest of China. In our greenhouse study, the metal resistance determinant in rhizobia was proved to be required for the functional symbiosis formation in the stress of heavy metals. In M. amorphae, chrA has been founded as a necessity for the formation of active nodules in the root of R. pseudoacacia at 25 mg/kg chromate stress, maintaining a regular expression level of leghemoglobin in the nodules and attaining a relative higher nitrogen accumulation than that of the △ chrA inoculation and un-inoculated control (unpublished). Similar results have also been observed in the nodules of inoculated Lentil, which attains a higher shoot N content and leghemoglobin content than the un-inoculated plants, but no results for the metal sensitive inoculated treatment (Wani et al., 2008c). Regarding M. lupulina, with similar nodulation frequency, there's no significant difference on the dry weight inoculated either with the S. meliloti parent or mutant strain (Li et al., 2014). Interestingly, S. meliloti could produce IAA under copper stressed conditions, with lower IAA production level achieved by copper sensitive mutants than that of the wild type strain (Li et al., 2014). Similarly, studies on the IAA production by A. tumefaciens found that IAA amount decreased with the increasing concentration of
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P. aeruginosa (AEQ93501). The broadly distributed and highly conserved motifs “FGGP” and “PGP” of these predicted ChrA proteins suggest a closely evolutionary relationship with other bacteria, indicating an ancient origin of the CHR family. Chromate may also be reduced in higher plants to Cr(III) by Cr reductases, even though no discrete enzymes have been identified (Lytle et al., 1998). There is, however, no such gene encoding a possible chromate reductase in the three PGRB strains. Thus, these three isolates may detoxify chromate mainly via the ATP-catalyzed ChrA efflux process.
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To sum up, zinc resistance in the three strains was realized via the cooperation the CDF family transporter and unspecific P-type ATP-catalyzed efflux process.
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Please cite this article as: Xie, P., et al., Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China, J. Environ. Sci. (2014), http://dx.doi.org/10.1016/j.jes.2014.07.017
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copper and zinc in the medium. R. pseudoacacia growth was much more greatly enhanced by inoculation of IAA production 342 strain A. tumefaciens compared to that of zinc sensitive mutant 343 (Hao et al., 2012a). Based on these findings, it can only be 344 concluded that the metal resistant genes may interfere with the 345 production of phytohorme of these two PGPBs via metal 346 homeostasis regulation. However, it's still needed to elucidate 347 how these metal resistance determinants interfere with the 348 nodulation and nitrogen fixation, the IAA synthesis, even some 349 un-covered but ion-mediated metabolic pathway. 350 Considering the advantages of using legumes as a resource 351 for phytoremediation (or phytostabilization), metal accumu352 lation capability of the legume plants inoculated by metal 353 resistant PGPB were analyzed. Actually, as the increasing of 354 metal concentrations in soil, the ion content in the tissues of 355 legume plants. While, R. pseudoacacia plants infected with 356 M. amorphae CCNWGS0123 led to a higher increment of 357 chromium content in the roots, but infection with the ΔchrA 358 mutant did not (unpublished). Regarding M. lupulina, plants 359 inoculated with copper-sensitive mutant accumulated much 360 less the metal in the tissues, compared to the plants inoculated 361 Q60 with S. meliloti CCNWSX0020 (Li et al., 2014). Similarly, Lupinus 362 albus inoculated with Bradyrhizobium L-7AH (metal resistant 363 strain) was able to accumulate large amounts of Hg in their 364 roots (Quiñones et al., 2013). Under metal stressed conditions, 365 this phenomenon that was explained by a greater biomass of 366 legumes was acquired via the inoculation with metal resistant 367 bacteria, but metal concentrations were redistributed among 368 plant tissues (Hao et al., 2014). It exactly indicates that 369 this legume–microbe system is suitable for the usage of 370 phytoremediation of metal contaminated soil, especially in 371 mine tailing regions.
contents (essential metals such as Zn, Cu, Fe, Mn, Co, as well as Ni; toxic heavy metals such as Cd and Cr) and metal toxicity of legumes, compared to that in other grains like rice, wheat, and maize. With a potential metal resistance and homeostasis network of the three metal tolerant PGPBs, the interaction of these microbes and their host plant symbiosis with respect to trace element homeostasis could be explored in several fields. Going in a top-down approach from an agricultural ecosystem to the sub-cellular level, it's necessary: (1) to understand the metal composition of the respective soil and how this influences the metal–microbe–plant interaction; (2) to metal resistance and homeostasis in the plant and the interacting bacterium; and (3) to how the various metal resistance determinants influence the metal distribution and interplay of in the plant and bacterial cell. This is especially true for soils that are in the mine tailing and contaminated with transition metals (Chen et al., 2012; Hanikenne and Nouet, 2011). Certainly, to better understand the complicated metal homeostasis of the microbe–plant symbiosis, from trace metal uptake to transport, from accumulation to detoxification, and from plant growth promotion to inhibition of rhizobium, further studies are needed both at biochemical and molecular levels. Molecular-genetic and physic-chem techniques will be applied to deeply study the interplay of the three PGRBs and their hosts.
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In the view of the whole genomic picture of the three metal resistant PGPB, M. amorphae contains protein members from P-type ATPase, CDF, HupE/UreJ and CHR family, which are expected to be involved in copper, zinc, nickel as well as chromate resistance and homeostasis. While, S. meliloti and A. tumefaciens exhibit a much more competent trafficking and detoxification network for copper and zinc homeostasis, respectively. Simon and Phung (1996) assumed that microbial metal resistance systems arose shortly after prokaryote life started. In fact, the resistance systems have been found in essentially all bacterial types, even though not precisely the same due to the substrate specific selection and environment adaptation (Nies, 2003). Lakzian et al. (2007) demonstrated that elevated levels of heavy metals induced the natural plasmid transferring between rhizobia. This study plays a crucial role in conferring enhanced and diversified metal tolerance of these dominant microbes in the mine tailings in Northwest of China. Legumes are such an important candidate that is 3rd largest family of flowering plants, widely existing in the forms of vegetable, oilseed, medicinal crops, animal feed (Ahmad et al., 2012). As a complement of cereals, it enters the human food chain and agriculture system directly or indirectly. However, little work has been performed on the determination of metal
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This work systematically presented the putative metal resistance determinant in the three PGPBs, containing transporter members from P-type ATPase, CDF, HupE/UreJ and CHR family involved in copper, zinc, nickel as well as chromate resistance. It was a very competent and intricate multi-metal homeostasis network, ensuring the survival capability of these isolates in the heavy polluted ecosystems. Together with their hosts, the M. amophae/A. tumefaciens–R. pseudoacacia, M. lupulina–S. meliloti symbiosis provides a potential tool for metal removal and soil fertility restoration in the metal tailing area. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jes.2014.07.017.
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This work was supported by the National Science Foundation of China (Nos. 31125007, 31370142), the National High Technology Research and Development Program (863) of China (No. 2012AA101402).
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