Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars (Populus alba L.)

Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white... Annals of Microbiology, 59 (1) 17-23 (2009) Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars (Populus alba L.) 1* 1 2 1 Alma BALESTRAZZI , Martina BONADEI , Emanuele QUATTRINI , Daniela CARBONERA 1 2 Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia; C.E.T.A.S., Università degli Studi di Milano, via Emilia, 26838 Tavazzano, Lodi, Italy Received 1 September 2008 / Accepted 17 December 2008 Abstract - The occurrence of multiple metal-resistance was assessed in two bacterial collections, named Herbicide Resistant Bacteria (HRB) and Nuclease-Producing Bacteria (NPB) respectively, consisting of 15 and 11 isolates obtained from a loamy sand cultivated with transgenic white poplars (Populus alba L., cv ‘Villafranca’) engineered for herbicide resistance. A third collection of 11 bacterial isolates, named Leaf-Associated Bacteria (LAB), obtained from the leaves of transgenic white poplars expressing the StSy gene for resveratrol production and from untransformed plants was evaluated. Resistance to Cd, Co, Cu, Pb and Zn was tested. As for the HRB collection, nine different phenotypes were monitored, which included tetra-, tri- and double-resistance. Tri- and double-metal resistance occurred also within the NPB and LAB collections. In both cases five different phenotypes were recovered. An additional investigation was carried out on the HRB-1c isolate, resistant to Cd, Co, Pb and Zn, which was previously demonstrated to produce indoleacetic acid, a plant- growth-promoting trait. Colorimetric assays, performed on the cell-depleted medium of HRB-1c liquid cultures grown in presence of heavy metals, confirmed that this trait was not affected. A 19-kb plasmid, possibly involved in the maintenance of the multiple metal- resistant phenotype, was detected in the HRB-1c cells. Key words: leaf-associated bacteria; multiple metal-resistance; transgenic poplar. INTRODUCTION potential in phytoremediation (Peuke and Rennenberg, 2006). Thus, the availability of bacterial isolates able to establish Nowadays heavy metal pollution in soils represents a severe optimal tree/microbe combinations is considered as a valu- problem for environmental and human health and, unfor- able tool to enhance the phytoremediation potential of elite tunately, remediation technologies still require consistent clones. From this point of view, the white poplar (Populus alba improvement (Lone et al., 2008; Mendez and Maier, 2008). L.) cultivar ‘Villafranca’ used in this work represents an ideal Although phytoremediation is currently considered a promising system, due to the high biomass production and resprouting strategy (Lone et al., 2008), a further optimization might be ability (Confalonieri et al., 2000). In recent years, the same achieved by exploiting the beneficial effects of plant-associated cultivar has been modified by gene transfer in order to acquire bacteria (Jing et al., 2007). Microorganisms can enhance the several agronomically relevant traits (Giorcelli et al., 2004; remediation ability of plants, reduce the phytotoxicity of pol- Zelasco et al., 2006; Balestrazzi et al., 2006), tested with luted soils and participate in heavy metal mobilization/immo- innovative marker-free gene-transfer technologies (Zelasco et bilization (Siciliano et al., 2001; Jing et al., 2007; Kuffner et al., 2007) and recently utilized for phytoremediation purposes al., 2008). (Castiglione et al., 2007; Lingua et al., 2008). Multiple metal-resistance, widespread in rhizobacteria A study on the environmental impact of transgenic white (Diaz-Ravina et al., 1994; Ryan et al., 2008), involves differ- poplars belonging to the ‘Villafranca’ cultivar has been also car- ent mechanisms such as enzymatic detoxification of the metal, ried out, focusing at the soil level. Such an investigation was binding of the metal to cell wall and to other specific cell com- possible since a greenhouse trial was established using two dif- ponents, blocking of metal uptake by cells and metal extrusion ferent classes of genetically modified (GM) white poplars: two by means of molecular pumps (Mergeay, 1991; Silver, 1992; transgenic lines expressing the bar gene from Streptomyces Nies, 1999). hygroscopicus, encoding an acetyltransferase able to inacti- To date, fast growing trees with high transpiration rates, vate phosphinothricin (PPT, the active component of Basta®) such as poplar, represent a suitable system with a great (Confalonieri et al., 2000) and two transgenic lines express- ing the StSy gene, from Vitis vinifera L., encoding stilbene synthase required for resveratrol biosynthesis (Giorcelli et al., * Corresponding Author. Phone: +39 0382 985435; 2004). Fax: +39 0382 528496; E-mail: almbal04@unipv.it 18 A. BALESTRAZZI et al. Two collections consisting of bacterial isolates obtained from line, leaves were collected from three different plants. Isolation the loamy sand cultivated with GM white poplars engineered of epiphytes was carried out on PCA medium as previously for herbicide tolerance are currently available. The Herbicide reported. The same leaves were subjected to surface sterilization Resistant Bacteria (HRB) and Nuclease-Producing Bacteria (NPB) by shaking in 2% NaClO for 10 min, then washed five times with collections have been already characterized in previous works sterile distilled water. Leaves were then grounded with a pestel in (Balestrazzi et al., 2007, 2008). small mortars containing 0.85% NaCl. Extracts were transferred The HRB collection, isolated on selective medium containing to sterile tubes, debris were removed by centrifugation (1500 PPT consisted mainly of Pseudomonas and Bacillus species and rpm, 5 min) and the liquid supernatant was treated as previously some of the tested HRB isolates showed useful properties for described in order to isolate the endophytic population. biotechnological and agronomical applications, such as swarm- All the HRB, NPB and LAB isolates examined in this study were ing motility and indoleacetic acid production (Balestrazzi et al., maintained at -70 °C in vials containing liquid LB medium sup- 2008). The NPB collection, isolated on a selective medium able plied with 50% glycerol. Aliquots (100 μl) were transferred from to reveal the presence extracellular DNase activity, included the stock vials onto Petri plates containing fresh Luria Bertani (LB) mainly Bacillus species and a few members of other gen- medium and bacteria were grown for two days at 28 °C. era, such as Brevibacillus, Microbacterium, Pseudomonas and Stenotrophomonas (Balestrazzi et al., 2007). Heavy metal-resistance. Resistance to heavy metals was eval- Besides this, a third collection, named Leaf-Associated uated on solid LB medium supplemented with 0.25 mM CdSO , Bacteria (LAB), has been produced starting from the leaf tissues 0.3 mM Co(NO ) , 10 mM CuCl , 50 mM PbCl and 3 mM ZnSO 3 2 2 2 4 of transgenic white poplars expressing the StSy gene for res- (Sigma-Aldrich). The salts were all dissolved in distilled water, veratrol synthesis (Balestrazzi et al., unpublished results). The sterilized by membrane filtration (Millipore Corporation, pore size LAB collection is made of leaf epiphytic and endophytic bacteria, 0.45 μm) and added to previously sterilized LB medium. The belonging to the Bacillus genus, which have been extensively bacterial isolates were transferred onto the selective plates by characterized for their tolerance to oxidative stress and to the means of replica plating. For each isolate, the ability to withstand antioxidant compound resveratrol. a single heavy metal was first tested. Subsequently, the multiple Endophytic bacteria are considered promising tools in the metal-resistant phenotypes were further assessed by combining field of bacteria-assisted phytoremediation and a deeper knowl- all the heavy metals in the same Petri plate. Bacterial growth was edge of heavy metal-resistant endophytic bacteria represents evaluated after incubation for 2 days at 28 °C. For each isolate, an essential prerequisite for effective phytoremediation of heavy three independent experiments with three replicated samples for metal-contaminated soils (Siciliano et al., 2001). each treatment were carried out. The present work reports on the occurrence of multiple metal-resistance in the HRB, NPB and LAB collections. This inves- Indoleacetic acid (IAA) production. IAA production was tigation will help identifying novel combinations of plant-growth measured as follows: 4-day-old cultures of the HRB-1c isolate promoting traits and multiple metal-resistant phenotypes for grown in liquid LB medium in presence/absence of heavy metals bacteria-assisted phytoremediation. were collected by centrifugation; aliquots (1 ml) of cell-depleted medium were mixed with 4 ml of Salkowsky’s reagent (150 ml of concentrated H SO , 250 ml of distilled H O, 7.5 ml of 0.5 2 4 2 -1 MATERIAL AND METHODS mol l FeCl .6H O) (Gordon and Weber, 1951) and incubated 3 2 at room temperature for 20 min. OD was measured and IAA Bacterial isolates. The HRB collection used in this study was concentrations were calculated by comparison with a standard recovered from an agricultural soil cultivated with transgenic curve, obtained using purified IAA (0.5, 1.0, 2.5, 5.0, 10.0 and -1 white poplars (Populus alba L. cv ‘Villafranca’) engineered with 20.0 μg ml ; Duchefa Biochemicals). Uninoculated medium the bar gene for herbicide tolerance as described by Balestrazzi with the reagent added was used as control. Three independent et al. (2008). Briefly, soil samples (1 g) were resuspended in 10 experiments, with three replicated samples for each treatment, ml of 0.85% NaCl and maintained under constant shaking (200 were carried out. rpm) for 30 min. The soil suspensions were serially diluted and plated onto Plate Count agar (PCA, Oxoid) supplemented with Extraction of plasmid DNA. Exponentially growing HRB-1c liquid -1 cycloheximide (100 mg ml , Duchefa Biochemicals) and phos- cultures incubated in LB medium supplemented with each single -1 phinothricin (PPT, 400 mg ml ). Plates were incubated at 28 °C metal (0.25 mM CdSO , 0.3 mM Co(NO ) , 50 mM PbCl and 3 4 3 2 2 for two days. mM ZnSO ) were utilized. The HRB-1c cells were also exposed As for the NPB collections, the same procedure was followed, simultaneously to all the toxic metals while, as control, they were except for the fact that the soil suspensions, serially diluted, grown in absence of pollutants. Heat was used as a plasmid-curing TM were plated onto Difco DNase Test Agar with Methylgreen agent (Gonzalez and Carlton, 1984). An exponentially grown HRB- (DGM Medium, Becton, Dickinson and Company Sparks, USA) 1c culture was used to inoculate pre-warmed LB medium at 43 °C. (Balestrazzi et al., 2007). The samples were then incubated at the same temperature for 24 The LAB collection, including epiphytic and endophytic h. The cultures were spread on agar plates containing the above bacteria associated with the leaves of transgenic white pop- reported heavy metals. Those colonies lacking metal tolerance were lars expressing the StSy gene for resveratrol production, was selected for plasmid extraction. To this purpose the plasmid-cured obtained from the GM lines 5EAC1 and 12EAC1 and from the HRB-1c cells were grown as previously described. Cell growth was untransformed line as previously reported. Leaves were collected monitored by measuring the optical density (OD ). Bacterial cells from two-year old white poplars during the vegetative growth were collected by centrifugation and plasmid DNA was extracted season (summer). Leaves were transferred to sterile tubes con- using the QIAGEN Plasmid Maxi Kit, according to the manufac- taining 5 ml of 0.85% NaCl and incubated at room temperature turer’s instructions. Conventional agarose gel electrophoresis was for 30 min, under constant shaking (200 rpm) in order to remove carried out with 0.8% (w/v) agarose (Duchefa Biochemicals), using the epiphytic bacteria living on the leaf surface. For each poplar the standard procedure described by Sambrook et al. (1989). Ann. Microbiol., 59 (1), 17-23 (2009) 19 TABLE 1 - Heavy-metal resistant phenotypes detected in the Herbicide Resistant Bacteria (HRB) collection a b Genus HRB isolate Multi heavy-metal resistance Cd Co Cu Pb Zn Pseudomonas HRB-1 + + - - + HRB-2 + + - - + HRB-3 + + - - + HRB-4 - + + - - HRB-5 + - + - - HRB-6 + + - - - HRB-7 ++ - + + HRB-8 ++ - + + HRB-9 + + - + + HRB-10 + + - + - HRB-11 - + - + - HRB-12 + - - + - Bacillus HRB-1a + + - + - HRB-1b - + + - - HRB-1c + + - + + For each HRB isolate, the genus has been determined in a previous work (Balestrazzi et al., 2008). Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM), CuCl (10 mM), PbCl (50 mM) and ZnSO 4 3 2 2 2 4 (3 mM). RESULTS Co and Pb. Finally, the HRB-1a isolate was able to survive Cd, Co and Pb. The double-resistant phenotype was common to five Occurrence of multiple metal-resistant phenotypes in the HRB isolates: HRB-4 (Co, Cu), HRB-5 (Cd, Cu), HRB-6 (Cd, Co), HRB collection HRB-11 (Co, Pb) and HRB-12 (Cd, Pb). The HRB isolates (Table 1) were grown in the presence of toxic metals supplied in the form of CdSO , Co(NO ) , CuCl , PbCl Occurrence of multiple metal-resistant phenotypes in the 4 3 2 2 2 and ZnSO . In a preliminary experiment, different doses for NPB collection each elemental pollutant were used, based on the current lit- As previously reported for the HRB collection, the NPB isolates erature (data not shown). The results reported in Table 1 refer (Table 2) were grown in the presence of different metals sup- to the highest concentration of heavy metal that allowed the plied in the form of CdSO , Co(NO ) , CuCl , PbCl and ZnSO . 4 3 2 2 2 4 growth of HRB isolates after two days. All the isolates revealed Results from these experiments, described in Table 2, represent multiple-resistant phenotypes which survived the following the highest heavy metal concentration that allowed growth after doses: 0.25 mM CdSO , 0.3 mM Co(NO ) , 10 mM CuCl , 50 two days. All the NPB isolates showed resistance to cobalt since 4 3 2 2 mM PbCl and 3.0 mM ZnSO . Nine different phenotypes were they were able to survive 0.3 mM Co(NO ) . The tri-resistant 2 4 3 2 monitored. As shown in Table 1, four HRB isolates out of 15 phenotype was found in the case of NPB-2, NPB-3 (Co, Pb, Zn) showed tetra-resistance. The HRB-7, HRB-8, HRB-9 and HRB- and NPB-11 (Cd, Co, Pb) isolates. A variegated range of double- 1c isolates could survive Cd, Co, Pb and Zn. Another group of resistant NPB isolates was observed: NPB-1, NPB-4, NPB-6, HRB isolates was characterized by tri-resistance. This included NPB-8 and NPB-10 (Co, Pb), HRB-7 and HRB-9 (Co, Zn), HRB-5 the HRB-1, HRB-2 and HRB-3 isolates which showed resistance (Co, Cu). to Cd, Co and Zn while the HRB-10 isolate could withstand Cd, TABLE 2 - Heavy-metal resistant phenotypes of the Nuclease-Producing Bacteria (NPB) collection a b Genus NPB isolate Multi heavy-metal resistance Cd Co Cu Pb Zn Bacillus NPB-1 - + - + - NPB-2 - + - + + NPB-3 - + - + + NPB-4 - + - + - NPB-5 - + + - - NPB-6 - + - + - Brevibacillus NPB-7 -+ - - + NPB-8 - +-+- Microbacterium NPB-9 - + - - + Pseudomonas NPB-10 - + - + - Stenotrophomonas NPB-11 + + - + - For each NPB isolate, the genus has been determined in a previous work (Balestrazzi et al., 2007). Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM) and CuCl (10 mM), PbCl (50 mM) and 4 3 2 2 2 ZnSO (3 mM). 4 20 A. BALESTRAZZI et al. TABLE 3 - Heavy-metal resistant phenotypes in the Leaf Associated Bacteria (LAB) collection. Epiphytic and endophytic bacteria were isolated from the leaves of the GM lines 5EAC1 and 12EAC1 and untransformed (CTRL) white poplars. All the LAB isolates are members of the genus Bacillus a b Poplar line Leaf-associated bacteria Multi heavy-metal resistance Cd Co Cu Pb Zn CTRL EP-C-1 - + + + - EP-C-2 - + - + + EN-C-1 - + - + + 5EAC1 EP-5-1 - + - - + EP-5-2 - + + - - EP-5-3 - + - + - EN-5-1 -+ + - - EN-5-2 -+ - - + 12EAC1 EP-12-1 - + - + - EP-12-2 - + - + - EN-12-2 - + + + - EP: epiphytic, EN: endophytic. Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM) and CuCl (10 mM), PbCl (50 mM) and 4 3 2 2 2 ZnSO (3 mM). Occurrence of multiple heavy metal-resistant phenotypes shown in Table 4. The IAA content in the cell-depleted medium -1 in the LAB collection of 4-day-old HRB-1c liquid cultures was 21.35 ± 0.79 μg ml The leaf-associated bacteria (Table 3) were tested for their ability (untreated control). In presence of heavy metals the rate of IAA to grow in the presence of different metals which were supplied production was always reduced. When single metals were sup- as CdSO (0.25 mM), Co(NO ) (0.3 mM), CuCl (10 mM), PbCl plied, the lowest IAA amount was recorded for Pb and Co (16.51 4 3 2 2 2 -1 (50 mM) and ZnSO (3.0 mM). Also in this case, results shown p 3.72 and 16.97 p 2.15 μg ml , respectively). Sligthly higher in Table 3, refer to the highest concentration of toxic metal that was the production in cultures containing Zn, 18.30 p 1.43 μg -1 allowed growth after two days. The LAB collection included iso- ml . In presence of cadmium, IAA production corresponded to -1 lates from untransformed (CTRL) and GM leaf tissues (5EAC1 20.14 p 1.60 μg ml . Finally, when all the four heavy metals and 12EAC1). Tri-resistance (Co, Pb, Zn) was recovered in two (Cd, Co, Pb, Zn) were added simultaneously to the medium, the -1 isolates deriving from untransformed white poplars, namely the resulting IAA biosynthesis, 14.65 p 3.27 μg ml , was significantly EP-C-2 and EN-C-1 and in one of the isolates obtained from the affected. GM line 12EAC1 (EN-12-2; Co, Cu, Pb). The double-resistant phenotypes occurred in all the LAB isolates associated with the Plasmid profile of HRB-1c isolate 5EAC1 GM line: isolates EP-5-1 and EN-5-2 showed resistance to In the attempt to assess the molecular basis of the tetra- Co and Zn while isolates EP-5-2 and EN-5-1 were resistant to Co resistant phenotype detected in the HRB-1c isolate, plasmid and Cu. Finally, the double-resistance to Co and Pb was a feature DNA was extracted from exponentially growing liquid cultures of EP-5-3, EP-12-1 and EP-12-2. exposed to each single pollutant (Cd, Co, Pb and Zn) and to all of them simultaneously. Conventional agarose gel electrophoresis Indoleacetic acid production in presence of heavy metals revealed the occurrence of a large plasmid (19.0 kb in size) (Fig. Since in a previous work the HRB-1c isolate was found to be the 1, lane 1). The same plasmid was absent in heat-cured cells (Fig. most active IAA producer, additional experiments were carried 1, lane 2). Differently from the wild type cells, the heat-cured out in order to verify whether this specific trait was maintained cells were not able to withstand Cd, Co, Pb and Zn when supplied in the presence of heavy metals. Results from these analyses are separately and in combination (Table 5). TABLE 4 - Indoleacetic acid production by HRB-1c isolate in TABLE 5 - Growth ability of wild type and plasmid-cured HRB-1c presence and absence of heavy metals isolate in presence and absence of heavy metals Heavy metals HRB-1c Heavy metals IAA production -1 (mg ml ) Wild type Plasmid cured Untreated control 21.35 ± 0.79 Untreated control + + Cd 20.14 ± 1.60 Cd + - Co 16.97 ± 2.15 Co + - Pb 16.51 ± 3.72 Pb + - Zn 18.30 ± 1.43 Zn + - Cd, Co, Pb, Zn 14.65 p 3.27 Cd, Co, Pb, Zn + - a a Metals were supplied to LB medium as CdSO (0.25 mM), Metals were supplied to LB medium as CdSO (0.25 mM), 4 4 Co(NO ) (0.3 mM), PbCl (50 mM) and ZnSO (3 mM). Co(NO ) (0.3 mM), PbCl (50 mM) and ZnSO (3 mM). 3 2 2 4 3 2 2 4 Ann. Microbiol., 59 (1), 17-23 (2009) 21 available, a discussion on the possible differences between GM M 1 2 and untransformed trees, in terms of leaf-associated microorgan- isms would be premature. However, it is worth noting that the kb LAB isolates associated with the GM lines 5EAC1 and 12EAC1 revealed a significant degree of resistance to oxidative stress 21.2 conditions when exposed to hydrogen peroxide and UV-C radia- tion, respectively, compared to isolates derived from untrans- 5.0 formed leaves (Balestrazzi et al., unpublished results). It has been hypothesized that endophytic microorganisms 2.0 harbouring traits for the effective removal of pollutants can sig- nificantly help metal mobilization through the plant vascular sys- tem. This might be particularly relevant in poplar trees, where a prolonged time might be required in order to move contaminants 0.5 from roots to leaves. Notwithstanding the several studies carried out on soil bacteria-assisted phytoremediation (Zaidi et al., 2006; Dell’Amico et al., 2008), to date there is limited information on the potential of endophytic bacteria on the phytoremediation of heavy metal-contaminated soils. It has been suggested that the use of LB media supplement- FIG. 1 - Plasmid-profile of HRB-1c isolate. Line 1: wild type ed with heavy metals could not represent the optimal choice to cells, line 2: plasmid-cured cells, line M: Lambda DNA/ investigate the bacterial response, due to the possible chelation/ EcoRI + HindIII Marker 3 (M-Medical s.r.l.). precipitation reactions occurring in micronutrient-rich culture media. This approach has been however reported by different authors in the case of cadmium, lead, zinc and copper (Yoshida et DISCUSSION al., 1998; Shakoori and Muneer, 2002; Zhigang et al., 2006). Since the HRB-1c isolate represents the most intriguing prod- Aim of the present work was to fulfil the phenotypic characteriza- uct so far obtained from our investigation, further analyses were tion of two different collections of bacterial isolates deriving from carried out to gain information on the molecular determinants a loamy sand cultivated with transgenic white poplars expressing of its tetra-resistant phenotype. A single extrachromosomal the bar gene for herbicide tolerance (Confalonieri et al., 2000). A element was evidenced by conventional gel electrophoresis in third collection, including the epiphytic and endophytic bacteria the HRB-1c cells. The plasmid (19.0 kb in size) was removed isolated from the leaves of transgenic white poplars expressing by heat-mediated curing and resistance to Cd, Co, Pb and Zn the StSy gene for resveratrol production (Giorcelli et al., 2004; disappeared. The occurrence of larger plasmids, visible only Balestrazzi et al., unpublished results) was also examined. using more sophisticated electrophoretic approaches, cannot All the heavy metals investigated in the present work cause be excluded. In a recent work, Pereira et al. (2006) reported severe environmental pollution (Lone et al., 2008). When the that cadmium resistance was linked to high molecular weight HRB collection was analysed, resistance to several metal combi- plasmids while the czc cation efflux system, able to actively nations was evidenced. The widest range of resistance included extrude cadmium, zinc and cobalt from the cell is encoded by a four heavy metals (Cd, Co, Pb and Zn) and it was found also large plasmid (Nies, 1992). Moreover, plasmids which harbour in the HRB-1c isolate, belonging to the Bacillus genus. HRB- genes involved in biotransformation of lead have been detected 1c is characterized by traits of agronomical relevance, such as in Staphylococcus aureus and Alcaligenes eutrophus (Mergeay, a remarkable swarming ability, the consistent production of 1991). indoleacetic acid and the ability to stimulate the in vitro growth When considering the total number of isolates examined in of white poplar explants (Balestrazzi et al., 2008). It has been this study, resistance to cobalt showed the highest frequency, reported that Cd can suppress auxin production of plant-growth- since it was recovered in 94.59% of isolates. This trait is generally stimulating bacteria (Kamnev et al., 2005). The finding that the dependent on cobalt-transporting CPx-Type ATPases (Rutherford HRB-1c isolate could maintain IAA biosynthesis also in the pres- et al., 1995) and on cobalt efflux proteins (Thorgensen and ence of Cd and other heavy metals enhances the biotechnological Downs, 2007). Lead resistance occurred in 62.16% of the total value of this microorganism as a tool for bacteria-assisted phy- bacteria examined. It has been reported that, in some cases, toremediation. resistance to lead may be connected with production of extracel- Resistance to Co was a common feature of NPB isolates and lular polymers or with the intracellular sequestration of this metal it associated with resistance to Pb, Cu or Zn, thus producing by bacterial cells (Roane, 1999). different double-resistant phenotypes in isolates classified as In contrast, resistance to Cd was found only in 35.13% of Bacillus, Brevibacillus, Microbacterium and Pseudomonas. Tri- the isolates. Exposure to cadmium induces synthesis of proteins resistance, involving Co, Pb and Zn and also Cd, Co and Pb, was that export or chelate the heavy metal while the intracellular glu- detected in NPB isolates belonging to the genera Bacillus and tathione-mediated detoxification of Cd has been also described in Stenotrophomonas (Balestrazzi et al., 2007). the genus Rhizobium (Figueira et al., 2005). Finally, resistance to Multiple metal-resistance occurred in both endophytic and zinc and copper occurred with a frequency of 43.24 and 18.91%, epiphytic bacteria. Tri-resistance was observed for all LAB iso- respectively. lates deriving from the leaves of untransformed white poplar and Detailed molecular analyses of the HRB, NPB and LAB isolates in the case of the EN-12-2 associated with the GM line 12EAC1. will be required to better understand the cellular and molecular As for the GM line 5EAC1, all the tested isolates, epiphytic and mechanisms responsible for the variegated patterns of multiple endophytic, showed a double-resistant phenotype. Based on the metal-resistance so far described. Additional studies will help reported data and considering the limited number of LAB isolates distinguishing between the resistance mechanisms which rely 22 A. BALESTRAZZI et al. on chromosomal genes, plasmids or transposons and the ubiq- The investigation focused on the capacity of HRB, NPB and uitary metal tolerance ascribed to general metabolic responses. LAB isolates to withstand significant heavy metal concentra- Mechanisms responsible for metal resistance might include tions. This ability would suggest for potential applications of such exclusion by permeability barrier, intra- and extracellular seques- bacteria in the field of in situ bioremediation of polluted environ- tration, efflux pumps, enzymatic detoxification, proteome and ments. Specific isolates with multiple metal-resistance might be metabolome changes (Silver, 1992; Nies, 1999; Baker-Austin et useful to target different elemental pollutants at contaminated sites. Furthermore, the ability to interact with perennial trees, al., 2006). Several studies have documented the predominance of e.g. those included in the genus Populus, might help to obtain Gram-negative bacteria in metal-contaminated soils and this novel plant-microbe remediation systems. finding might be explained by the fact that the Gram-negative Acknowledgments cell wall is a more efficient barrier to toxic metals than the This research was supported by a grant from Regione Lombardia Gram-positive one (Duxbury and Bicknell, 1983). In accord- (Divisione Generale Agricoltura). M.B. received a doctoral fellow- ance with this hypothesis, although most of the environmental ship from Regione Lombardia. isolates (62.1%) examined in this study were Gram-positive, the tetra-resistant phenotypes were more frequent among the Gram-negative bacteria belonging to the genus Pseudomonas. REFERENCES The wide range of heavy metal resistant phenotypes rou- tinely isolated from soil bacteria might be related to the common Baker-Austin C., Wright M.S., Stepanauskas R., McArthur J.V. agricultural practices which rely upon the use of metal-containing (2006). Co-selection of antibiotic and metal resistance. products, such as fertilizers, fungicides, pesticides and her- Trends Microbiol., 14: 176-182. bicides, routinely adopted to enhance crop production (He et al., 2005). It is worth noting that both the herbicide-tolerant Balestrazzi A., Allegro G., Confalonieri M. (2006). Genetically and resveratrol-producing white poplars grown in greenhouses modified trees expressing genes for insect pest resistance. underwent intensive fertilization treatments and nutrients solu- In: Fladung M., Ewald D., Eds, Tree Transgenesis: Recent tions enriched in microelements were supplied to support growth Developments, Springer-Verlag Berlin Heidelberg, pp. 253- (Balestrazzi et al., 2008). The heavy metals tested in this work 273. include micronutrients such as Co, Cu and Zn which are essen- Balestrazzi A., Bonadei M., Carbonera D. (2007). Nuclease- tial components of redox processes and cofactors of relevant producing bacteria in soil cultivated with herbicide resistant enzymes (O’Halloran, 1993). It is known that bacteria possess transgenic white poplars. Ann. Microbiol., 57: 531- 536. copper resistance operons which display metal-responsive tran- Balestrazzi A., Bonadei M., Zelasco S., Quattrini E., Calvio C., scriptional regulation with membrane-spanning proteins involved Galizzi A., Carbonera D. (2008). Recovery of useful traits in monitoring changes in copper concentrations in periplasm from isolates inhabiting an agricultural soil cultivated with (O’Halloran, 1993). On the other hand, zinc is found in enzymes herbicide-resistant poplars. Can. J. Microbiol., 54: 201-208. involved in transcription and in transcription factors respon- Castiglione S., Franchin C., Fossati T., Lingua G., Torrigiani P., sive to a variety of intra- and inter-cellular signals (O’Halloran, Biondi S. (2007). High zinc concentrations reduce rooting 1993). Cadmium is the only non essential element evaluated in capacity and alter metallothionein gene expression in white the present work. Resistance to essential elements is usually poplar (Populus alba cv. Villafranca). Chemosphere, 67: chromosome-based while the resistance to non essential trace 1117-1126. elements is associated with plasmids and transposons (Silver, Collins Y.E., Stotzky G. (1989). Factors affecting the toxicity of 1992). This is in accordance with the reported data linking the heavy metals to microbes. In: Beveridge T.J., Doyle R.J., Eds, resistance to cadmium and the 19-kb plasmid detected in HRB-1c cells. Within this context the correlation between this extracel- Metal Ions and Bacteria, John Wiley and Sons Inc., New York, pp. 31-90. lular element and resistance to Co, Pb and Zn remains difficult to explain. However, in case of plasmid-determined copper resist- Confalonieri M., Belenghi B., Balestrazzi A., Negri S., Facciotto G., ance mechanisms involving bioaccumulation, sequestration and Schenone G., DelleDonne M. (2000). Transformation of elite efflux have been documented (Silver, 1992). white poplar (P. alba) cv Villafranca and evaluation of herbi- Finally, it has been reported that heavy metal toxicity might cide resistance. Plant Cell Rep., 19: 978-982. be lowered in soils with high clay, since the charged clay parti- Dell’Amico E., Mazzocchi M., Cavalca L., Allievi L., Andreoni V. cles limit metal availability by sequestration (Collins and Stotzky, (2008). Assessment of bacterial community structure in 1989). The medium-textured loamy sand utilized for white poplar a long-term copper-polluted ex-vineyard soil. Microbiol. cultivation in greenhouse was characterized by a reduced clay Res.,163: 671-683. content (5.62%) (Balestrazzi et al., 2007, 2008) and this might Diaz-Ravina M., Baath E., Frostegard A. (1994). Multiple heavy have contributed to the high frequency of multiple metal-resist- metal tolerance of soil bacterial communities and its meas- ance detected in the bacterial isolates. urement by a thymidine incorporation technique. Appl. The response of HRB, NPB and LAB isolates to a wider range Environ. Microbiol., 60: 2238-2247. of trace elements, including As, Se and Hg, will possibly reveal Duxbury T., Bicknell B. (1983). Metal-tolerant bacterial popu- additional resistance traits useful for waste bioremediation or lations from natural and metal polluted soils. Soil Biol. biometallurgy. To date, microbial studies are required to identify Biochem., 9: 105-112. novel soil culturable microorganisms able to support the phy- toremediation performace of specific plant systems (Lone et al., Figueira E.M.A.P., Lima A.I.G., Pereira S.I.A. (2005). Cadmium 2008). The present work presents a list of novel environmental tolerance plasticity in Rhizobium leguminosarum bv. viciae: isolates with multiple metal-resistance which will be useful in glutathione as a detoxifying agent. Can. J. Microbiol., 51: future studies on bacteria-assisted phytoremediation with trees. 7-14. Ann. Microbiol., 59 (1), 17-23 (2009) 23 Giorcelli A., Sparvoli F., Mattivi F., Balestrazzi A., Tava A., Ewald D., Eds, Tree Transgenesis: Recent Developments, Vrhovsek U., Bollini R., Confalonieri M. (2004). Expression of Springer-Verlag Berlin Heidelberg, pp. 253-273. stilbene synthase (StSy) gene from grapevine in transgenic Roane T.M. (1999). Lead resistance in two isolates from heavy white poplar results in high accumulation of the antioxidant metal-contaminated soils. Microb. Ecol., 37: 218-224. compounds resveratrol glucosides. Trans. Res., 13: 203- Rutherford J.C., Cavet J.S., Robinson N.J. (1995). Cobalt- dependent transcriptional switching by a dual-effector MerR- Gonzalez J.M.Jr, Carlton B.C. (1984). A large transmissable plas- like protein regulates a cobalt-exporting variant CPx-type mid is required for crystal toxin production in Bacillus thur- ATPase. J. Biol. Chem., 274: 25827-25832. ingiensis variety israelensis. Plasmid, 11: 28-38. Ryan R., Ryan D., Dowling D. (2008). Multiple metal resistant Gordon S.A., Weber R.P. (1951). Colorimetric estimation of transferable phenotypes in bacteria as indicators of soil con- indoleacetic acid. Plant Physiol., 26: 192-195. tamination with heavy metals. J. Soils Sedim., 5: 95-100. He Z.L., Yang X.E., Stoffella P.J. (2005). Trace elements in agr- Sambrook J., Fritsch E.F., Maniatis T. (1989). Molecular Cloning: A nd oecosystems and impacts on the environment. J. Trace Elem. Laboratory Manual, 2 edn., Cold Spring Harbor Laboratory Med. Biol., 19: 125-140. Press, Cold Spring Harbor, New York. Jing Y., He Z., Yang X. (2007). Role of soil rhizobacteria in phy- Shakoori A.R., Muneer B. (2002). Copper-resistant bacteria from toremediation of heavy metal contaminated soils. J. Zhejiang industrial effluents and their role in remediation of heavy Univ. Sci. B, 8: 192-207. metals in wastewater. Folia Microbiol., 47: 43-50. Kamnev A.A., Tugarova A.V., Antonyuk L.P., Tarantilis P.A., Siciliano S.D., Fortin N., Mihoc A., Wisse G., Labelle S., Beaumier Polissiou M.G., Gardiner P.H. (2005). Effects of heavy metals D., Ouellette D., Real R., Whyte L.G., Banks M.K., Schwab P., on plant-associated rhizobacteria: comparison of endophytic Lee K., Greer C.W. (2001). Selection of specific endophytic and non-endophytic strains of Azospirillum brasilense. J. bacterial genotypes by plants in response to soil contamina- Trace Elem. Med. Biol., 19: 91-95. tion. Appl. Environ. Microbiol., 67: 2496-2475. Kuffner M., Poschenrieder M., Wieshammer G., Gorfer M., Silver S. (1992). Plasmid-determined metal resistance mecha- Sessitsch A. (2008). Rhizosphere bacteria affect growth and nisms: range and overview. Plasmid, 27: 1-3. metal uptake of heavy metal accumulating willows. Plant Thorgensen M.P., Downs D. (2007). Cobalt targets multiple Soil, 304: 35-44. metabolic processes in Salmonella enterica. J. Bacteriol., Lingua G., Franchin C., Todeschini V., Castiglione S., Biondi S., 189: 7774-7781. Burlando B., Parravicini V., Torrigiani P., Berta G. (2008). Yoshida N., Murooka Y., Ogawa K. (1998). Heavy metal particle Arbuscolar mycorrhizal fungi differentially affect the response resistance in Thiobacillus intermedius 13-1 isolated from cor- to high zinc concentrations of two registered poplar clones, roded concrete. J. Ferment. Bioeng., 85: 630-633. Villafranca (Populus alba L.) and Jean Pourtet (Populus nigra Zaidi S., Usmani S., Singh B.R., Musarrat J. (2006). Significance L.). Environ. Pollut., 153: 137-147. of Bacillus subtilis strain SJ-101 as a bioinoculant for con- Lone M.I., He Z., Stoffella P.J., Yang X. (2008). Phytoremediation current plant growth promotion and nickel accumulation in of heavy metal polluted soils and water: progresses and per- Brassica juncea. Chemosphere, 64: 991-997. spectives. J. Zhejiang Univ. Sci. B, 9: 210-220. Zelasco S., Reggi S., Calligari P., Balestrazzi A., Bongiorni C., Mendez M.O., Maier R.M. (2008). Phytostabilization of mine tail- Quattrini E., Delia G., Bisoffi S., Fogher C., Confalonieri M. ings in arid and semiarid environments - an emerging reme- (2006). Expression of the Vitreoscilla hemoglobin (VHb)- diation technology. Environ. Health Perspect., 116: 278-283. encoding gene in transgenic white poplar: plant growth and biomass production, biochemical characterization and cell Mergeay M. (1991). Towards an understanding of the genetics of survival under submergence, oxidative and nitrosative stress bacterial metal resistance. Trends Biotechnol., 9: 17-24. conditions. Mol. Breed., 17: 201-216. Nies D.H. (1992). Resistance to cadmium, cobalt, zinc and nickel Zelasco S., Ressegotti V., Confalonieri M., Carbonera D., Calligari in microbes. Plasmid, 27: 17-28. P., Bonadei M., Bisoffi S., Yamada K., Balestrazzi A. (2007). Nies D.H. (1999). Microbial heavy metal resistance. Appl. Evaluation of MAT-vector system in white poplar (Populus Microbiol. Biotechnol., 51: 730-750. alba L.) and production of ipt marker-free transgenic plants O’Halloran T. (1993). Transition metals in control of gene expres- by ‘single-step transformation’. Plant Cell Tiss. Org. Cult., sion. Science, 261: 717-725. 91: 61-72. Pereira S.I., Lima A.I., Figueira E.M. (2006) Screening possible Zhigang A., Cuijie L., Yuangang Z., Yeljie D., Watcher A., Gromes mechanisms mediating cadmium resistance in Rhizobium R., Rausch T. (2006). Expression of BjMT2, a metallothionein leguminosarum bv. viciae isolated from contaminated portu- 2 from Brassica juncea, incresaes copper and cadmium toler- guese soils. Microb. Ecol., 52: 176-186. ance in Escherichia coli and Arabidopsis thaliana, but inhibits Peuke A.D., Rennenberg H. (2006). Heavy metal resistance and root elongation in Arabidopsis thaliana seedlings. J. Exp. Bot., phytoremediation with transgenic trees. In: Fladung M., 57: 3575-3582. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars (Populus alba L.)

Loading next page...
 
/lp/springer-journals/occurrence-of-multiple-metal-resistance-in-bacterial-isolates-pYYwPdsS69

References (52)

Publisher
Springer Journals
Copyright
Copyright © 2009 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/BF03175593
Publisher site
See Article on Publisher Site

Abstract

Annals of Microbiology, 59 (1) 17-23 (2009) Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars (Populus alba L.) 1* 1 2 1 Alma BALESTRAZZI , Martina BONADEI , Emanuele QUATTRINI , Daniela CARBONERA 1 2 Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia; C.E.T.A.S., Università degli Studi di Milano, via Emilia, 26838 Tavazzano, Lodi, Italy Received 1 September 2008 / Accepted 17 December 2008 Abstract - The occurrence of multiple metal-resistance was assessed in two bacterial collections, named Herbicide Resistant Bacteria (HRB) and Nuclease-Producing Bacteria (NPB) respectively, consisting of 15 and 11 isolates obtained from a loamy sand cultivated with transgenic white poplars (Populus alba L., cv ‘Villafranca’) engineered for herbicide resistance. A third collection of 11 bacterial isolates, named Leaf-Associated Bacteria (LAB), obtained from the leaves of transgenic white poplars expressing the StSy gene for resveratrol production and from untransformed plants was evaluated. Resistance to Cd, Co, Cu, Pb and Zn was tested. As for the HRB collection, nine different phenotypes were monitored, which included tetra-, tri- and double-resistance. Tri- and double-metal resistance occurred also within the NPB and LAB collections. In both cases five different phenotypes were recovered. An additional investigation was carried out on the HRB-1c isolate, resistant to Cd, Co, Pb and Zn, which was previously demonstrated to produce indoleacetic acid, a plant- growth-promoting trait. Colorimetric assays, performed on the cell-depleted medium of HRB-1c liquid cultures grown in presence of heavy metals, confirmed that this trait was not affected. A 19-kb plasmid, possibly involved in the maintenance of the multiple metal- resistant phenotype, was detected in the HRB-1c cells. Key words: leaf-associated bacteria; multiple metal-resistance; transgenic poplar. INTRODUCTION potential in phytoremediation (Peuke and Rennenberg, 2006). Thus, the availability of bacterial isolates able to establish Nowadays heavy metal pollution in soils represents a severe optimal tree/microbe combinations is considered as a valu- problem for environmental and human health and, unfor- able tool to enhance the phytoremediation potential of elite tunately, remediation technologies still require consistent clones. From this point of view, the white poplar (Populus alba improvement (Lone et al., 2008; Mendez and Maier, 2008). L.) cultivar ‘Villafranca’ used in this work represents an ideal Although phytoremediation is currently considered a promising system, due to the high biomass production and resprouting strategy (Lone et al., 2008), a further optimization might be ability (Confalonieri et al., 2000). In recent years, the same achieved by exploiting the beneficial effects of plant-associated cultivar has been modified by gene transfer in order to acquire bacteria (Jing et al., 2007). Microorganisms can enhance the several agronomically relevant traits (Giorcelli et al., 2004; remediation ability of plants, reduce the phytotoxicity of pol- Zelasco et al., 2006; Balestrazzi et al., 2006), tested with luted soils and participate in heavy metal mobilization/immo- innovative marker-free gene-transfer technologies (Zelasco et bilization (Siciliano et al., 2001; Jing et al., 2007; Kuffner et al., 2007) and recently utilized for phytoremediation purposes al., 2008). (Castiglione et al., 2007; Lingua et al., 2008). Multiple metal-resistance, widespread in rhizobacteria A study on the environmental impact of transgenic white (Diaz-Ravina et al., 1994; Ryan et al., 2008), involves differ- poplars belonging to the ‘Villafranca’ cultivar has been also car- ent mechanisms such as enzymatic detoxification of the metal, ried out, focusing at the soil level. Such an investigation was binding of the metal to cell wall and to other specific cell com- possible since a greenhouse trial was established using two dif- ponents, blocking of metal uptake by cells and metal extrusion ferent classes of genetically modified (GM) white poplars: two by means of molecular pumps (Mergeay, 1991; Silver, 1992; transgenic lines expressing the bar gene from Streptomyces Nies, 1999). hygroscopicus, encoding an acetyltransferase able to inacti- To date, fast growing trees with high transpiration rates, vate phosphinothricin (PPT, the active component of Basta®) such as poplar, represent a suitable system with a great (Confalonieri et al., 2000) and two transgenic lines express- ing the StSy gene, from Vitis vinifera L., encoding stilbene synthase required for resveratrol biosynthesis (Giorcelli et al., * Corresponding Author. Phone: +39 0382 985435; 2004). Fax: +39 0382 528496; E-mail: almbal04@unipv.it 18 A. BALESTRAZZI et al. Two collections consisting of bacterial isolates obtained from line, leaves were collected from three different plants. Isolation the loamy sand cultivated with GM white poplars engineered of epiphytes was carried out on PCA medium as previously for herbicide tolerance are currently available. The Herbicide reported. The same leaves were subjected to surface sterilization Resistant Bacteria (HRB) and Nuclease-Producing Bacteria (NPB) by shaking in 2% NaClO for 10 min, then washed five times with collections have been already characterized in previous works sterile distilled water. Leaves were then grounded with a pestel in (Balestrazzi et al., 2007, 2008). small mortars containing 0.85% NaCl. Extracts were transferred The HRB collection, isolated on selective medium containing to sterile tubes, debris were removed by centrifugation (1500 PPT consisted mainly of Pseudomonas and Bacillus species and rpm, 5 min) and the liquid supernatant was treated as previously some of the tested HRB isolates showed useful properties for described in order to isolate the endophytic population. biotechnological and agronomical applications, such as swarm- All the HRB, NPB and LAB isolates examined in this study were ing motility and indoleacetic acid production (Balestrazzi et al., maintained at -70 °C in vials containing liquid LB medium sup- 2008). The NPB collection, isolated on a selective medium able plied with 50% glycerol. Aliquots (100 μl) were transferred from to reveal the presence extracellular DNase activity, included the stock vials onto Petri plates containing fresh Luria Bertani (LB) mainly Bacillus species and a few members of other gen- medium and bacteria were grown for two days at 28 °C. era, such as Brevibacillus, Microbacterium, Pseudomonas and Stenotrophomonas (Balestrazzi et al., 2007). Heavy metal-resistance. Resistance to heavy metals was eval- Besides this, a third collection, named Leaf-Associated uated on solid LB medium supplemented with 0.25 mM CdSO , Bacteria (LAB), has been produced starting from the leaf tissues 0.3 mM Co(NO ) , 10 mM CuCl , 50 mM PbCl and 3 mM ZnSO 3 2 2 2 4 of transgenic white poplars expressing the StSy gene for res- (Sigma-Aldrich). The salts were all dissolved in distilled water, veratrol synthesis (Balestrazzi et al., unpublished results). The sterilized by membrane filtration (Millipore Corporation, pore size LAB collection is made of leaf epiphytic and endophytic bacteria, 0.45 μm) and added to previously sterilized LB medium. The belonging to the Bacillus genus, which have been extensively bacterial isolates were transferred onto the selective plates by characterized for their tolerance to oxidative stress and to the means of replica plating. For each isolate, the ability to withstand antioxidant compound resveratrol. a single heavy metal was first tested. Subsequently, the multiple Endophytic bacteria are considered promising tools in the metal-resistant phenotypes were further assessed by combining field of bacteria-assisted phytoremediation and a deeper knowl- all the heavy metals in the same Petri plate. Bacterial growth was edge of heavy metal-resistant endophytic bacteria represents evaluated after incubation for 2 days at 28 °C. For each isolate, an essential prerequisite for effective phytoremediation of heavy three independent experiments with three replicated samples for metal-contaminated soils (Siciliano et al., 2001). each treatment were carried out. The present work reports on the occurrence of multiple metal-resistance in the HRB, NPB and LAB collections. This inves- Indoleacetic acid (IAA) production. IAA production was tigation will help identifying novel combinations of plant-growth measured as follows: 4-day-old cultures of the HRB-1c isolate promoting traits and multiple metal-resistant phenotypes for grown in liquid LB medium in presence/absence of heavy metals bacteria-assisted phytoremediation. were collected by centrifugation; aliquots (1 ml) of cell-depleted medium were mixed with 4 ml of Salkowsky’s reagent (150 ml of concentrated H SO , 250 ml of distilled H O, 7.5 ml of 0.5 2 4 2 -1 MATERIAL AND METHODS mol l FeCl .6H O) (Gordon and Weber, 1951) and incubated 3 2 at room temperature for 20 min. OD was measured and IAA Bacterial isolates. The HRB collection used in this study was concentrations were calculated by comparison with a standard recovered from an agricultural soil cultivated with transgenic curve, obtained using purified IAA (0.5, 1.0, 2.5, 5.0, 10.0 and -1 white poplars (Populus alba L. cv ‘Villafranca’) engineered with 20.0 μg ml ; Duchefa Biochemicals). Uninoculated medium the bar gene for herbicide tolerance as described by Balestrazzi with the reagent added was used as control. Three independent et al. (2008). Briefly, soil samples (1 g) were resuspended in 10 experiments, with three replicated samples for each treatment, ml of 0.85% NaCl and maintained under constant shaking (200 were carried out. rpm) for 30 min. The soil suspensions were serially diluted and plated onto Plate Count agar (PCA, Oxoid) supplemented with Extraction of plasmid DNA. Exponentially growing HRB-1c liquid -1 cycloheximide (100 mg ml , Duchefa Biochemicals) and phos- cultures incubated in LB medium supplemented with each single -1 phinothricin (PPT, 400 mg ml ). Plates were incubated at 28 °C metal (0.25 mM CdSO , 0.3 mM Co(NO ) , 50 mM PbCl and 3 4 3 2 2 for two days. mM ZnSO ) were utilized. The HRB-1c cells were also exposed As for the NPB collections, the same procedure was followed, simultaneously to all the toxic metals while, as control, they were except for the fact that the soil suspensions, serially diluted, grown in absence of pollutants. Heat was used as a plasmid-curing TM were plated onto Difco DNase Test Agar with Methylgreen agent (Gonzalez and Carlton, 1984). An exponentially grown HRB- (DGM Medium, Becton, Dickinson and Company Sparks, USA) 1c culture was used to inoculate pre-warmed LB medium at 43 °C. (Balestrazzi et al., 2007). The samples were then incubated at the same temperature for 24 The LAB collection, including epiphytic and endophytic h. The cultures were spread on agar plates containing the above bacteria associated with the leaves of transgenic white pop- reported heavy metals. Those colonies lacking metal tolerance were lars expressing the StSy gene for resveratrol production, was selected for plasmid extraction. To this purpose the plasmid-cured obtained from the GM lines 5EAC1 and 12EAC1 and from the HRB-1c cells were grown as previously described. Cell growth was untransformed line as previously reported. Leaves were collected monitored by measuring the optical density (OD ). Bacterial cells from two-year old white poplars during the vegetative growth were collected by centrifugation and plasmid DNA was extracted season (summer). Leaves were transferred to sterile tubes con- using the QIAGEN Plasmid Maxi Kit, according to the manufac- taining 5 ml of 0.85% NaCl and incubated at room temperature turer’s instructions. Conventional agarose gel electrophoresis was for 30 min, under constant shaking (200 rpm) in order to remove carried out with 0.8% (w/v) agarose (Duchefa Biochemicals), using the epiphytic bacteria living on the leaf surface. For each poplar the standard procedure described by Sambrook et al. (1989). Ann. Microbiol., 59 (1), 17-23 (2009) 19 TABLE 1 - Heavy-metal resistant phenotypes detected in the Herbicide Resistant Bacteria (HRB) collection a b Genus HRB isolate Multi heavy-metal resistance Cd Co Cu Pb Zn Pseudomonas HRB-1 + + - - + HRB-2 + + - - + HRB-3 + + - - + HRB-4 - + + - - HRB-5 + - + - - HRB-6 + + - - - HRB-7 ++ - + + HRB-8 ++ - + + HRB-9 + + - + + HRB-10 + + - + - HRB-11 - + - + - HRB-12 + - - + - Bacillus HRB-1a + + - + - HRB-1b - + + - - HRB-1c + + - + + For each HRB isolate, the genus has been determined in a previous work (Balestrazzi et al., 2008). Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM), CuCl (10 mM), PbCl (50 mM) and ZnSO 4 3 2 2 2 4 (3 mM). RESULTS Co and Pb. Finally, the HRB-1a isolate was able to survive Cd, Co and Pb. The double-resistant phenotype was common to five Occurrence of multiple metal-resistant phenotypes in the HRB isolates: HRB-4 (Co, Cu), HRB-5 (Cd, Cu), HRB-6 (Cd, Co), HRB collection HRB-11 (Co, Pb) and HRB-12 (Cd, Pb). The HRB isolates (Table 1) were grown in the presence of toxic metals supplied in the form of CdSO , Co(NO ) , CuCl , PbCl Occurrence of multiple metal-resistant phenotypes in the 4 3 2 2 2 and ZnSO . In a preliminary experiment, different doses for NPB collection each elemental pollutant were used, based on the current lit- As previously reported for the HRB collection, the NPB isolates erature (data not shown). The results reported in Table 1 refer (Table 2) were grown in the presence of different metals sup- to the highest concentration of heavy metal that allowed the plied in the form of CdSO , Co(NO ) , CuCl , PbCl and ZnSO . 4 3 2 2 2 4 growth of HRB isolates after two days. All the isolates revealed Results from these experiments, described in Table 2, represent multiple-resistant phenotypes which survived the following the highest heavy metal concentration that allowed growth after doses: 0.25 mM CdSO , 0.3 mM Co(NO ) , 10 mM CuCl , 50 two days. All the NPB isolates showed resistance to cobalt since 4 3 2 2 mM PbCl and 3.0 mM ZnSO . Nine different phenotypes were they were able to survive 0.3 mM Co(NO ) . The tri-resistant 2 4 3 2 monitored. As shown in Table 1, four HRB isolates out of 15 phenotype was found in the case of NPB-2, NPB-3 (Co, Pb, Zn) showed tetra-resistance. The HRB-7, HRB-8, HRB-9 and HRB- and NPB-11 (Cd, Co, Pb) isolates. A variegated range of double- 1c isolates could survive Cd, Co, Pb and Zn. Another group of resistant NPB isolates was observed: NPB-1, NPB-4, NPB-6, HRB isolates was characterized by tri-resistance. This included NPB-8 and NPB-10 (Co, Pb), HRB-7 and HRB-9 (Co, Zn), HRB-5 the HRB-1, HRB-2 and HRB-3 isolates which showed resistance (Co, Cu). to Cd, Co and Zn while the HRB-10 isolate could withstand Cd, TABLE 2 - Heavy-metal resistant phenotypes of the Nuclease-Producing Bacteria (NPB) collection a b Genus NPB isolate Multi heavy-metal resistance Cd Co Cu Pb Zn Bacillus NPB-1 - + - + - NPB-2 - + - + + NPB-3 - + - + + NPB-4 - + - + - NPB-5 - + + - - NPB-6 - + - + - Brevibacillus NPB-7 -+ - - + NPB-8 - +-+- Microbacterium NPB-9 - + - - + Pseudomonas NPB-10 - + - + - Stenotrophomonas NPB-11 + + - + - For each NPB isolate, the genus has been determined in a previous work (Balestrazzi et al., 2007). Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM) and CuCl (10 mM), PbCl (50 mM) and 4 3 2 2 2 ZnSO (3 mM). 4 20 A. BALESTRAZZI et al. TABLE 3 - Heavy-metal resistant phenotypes in the Leaf Associated Bacteria (LAB) collection. Epiphytic and endophytic bacteria were isolated from the leaves of the GM lines 5EAC1 and 12EAC1 and untransformed (CTRL) white poplars. All the LAB isolates are members of the genus Bacillus a b Poplar line Leaf-associated bacteria Multi heavy-metal resistance Cd Co Cu Pb Zn CTRL EP-C-1 - + + + - EP-C-2 - + - + + EN-C-1 - + - + + 5EAC1 EP-5-1 - + - - + EP-5-2 - + + - - EP-5-3 - + - + - EN-5-1 -+ + - - EN-5-2 -+ - - + 12EAC1 EP-12-1 - + - + - EP-12-2 - + - + - EN-12-2 - + + + - EP: epiphytic, EN: endophytic. Metals were supplied to LB medium as CdSO (0.25 mM), Co(NO ) (0.3 mM) and CuCl (10 mM), PbCl (50 mM) and 4 3 2 2 2 ZnSO (3 mM). Occurrence of multiple heavy metal-resistant phenotypes shown in Table 4. The IAA content in the cell-depleted medium -1 in the LAB collection of 4-day-old HRB-1c liquid cultures was 21.35 ± 0.79 μg ml The leaf-associated bacteria (Table 3) were tested for their ability (untreated control). In presence of heavy metals the rate of IAA to grow in the presence of different metals which were supplied production was always reduced. When single metals were sup- as CdSO (0.25 mM), Co(NO ) (0.3 mM), CuCl (10 mM), PbCl plied, the lowest IAA amount was recorded for Pb and Co (16.51 4 3 2 2 2 -1 (50 mM) and ZnSO (3.0 mM). Also in this case, results shown p 3.72 and 16.97 p 2.15 μg ml , respectively). Sligthly higher in Table 3, refer to the highest concentration of toxic metal that was the production in cultures containing Zn, 18.30 p 1.43 μg -1 allowed growth after two days. The LAB collection included iso- ml . In presence of cadmium, IAA production corresponded to -1 lates from untransformed (CTRL) and GM leaf tissues (5EAC1 20.14 p 1.60 μg ml . Finally, when all the four heavy metals and 12EAC1). Tri-resistance (Co, Pb, Zn) was recovered in two (Cd, Co, Pb, Zn) were added simultaneously to the medium, the -1 isolates deriving from untransformed white poplars, namely the resulting IAA biosynthesis, 14.65 p 3.27 μg ml , was significantly EP-C-2 and EN-C-1 and in one of the isolates obtained from the affected. GM line 12EAC1 (EN-12-2; Co, Cu, Pb). The double-resistant phenotypes occurred in all the LAB isolates associated with the Plasmid profile of HRB-1c isolate 5EAC1 GM line: isolates EP-5-1 and EN-5-2 showed resistance to In the attempt to assess the molecular basis of the tetra- Co and Zn while isolates EP-5-2 and EN-5-1 were resistant to Co resistant phenotype detected in the HRB-1c isolate, plasmid and Cu. Finally, the double-resistance to Co and Pb was a feature DNA was extracted from exponentially growing liquid cultures of EP-5-3, EP-12-1 and EP-12-2. exposed to each single pollutant (Cd, Co, Pb and Zn) and to all of them simultaneously. Conventional agarose gel electrophoresis Indoleacetic acid production in presence of heavy metals revealed the occurrence of a large plasmid (19.0 kb in size) (Fig. Since in a previous work the HRB-1c isolate was found to be the 1, lane 1). The same plasmid was absent in heat-cured cells (Fig. most active IAA producer, additional experiments were carried 1, lane 2). Differently from the wild type cells, the heat-cured out in order to verify whether this specific trait was maintained cells were not able to withstand Cd, Co, Pb and Zn when supplied in the presence of heavy metals. Results from these analyses are separately and in combination (Table 5). TABLE 4 - Indoleacetic acid production by HRB-1c isolate in TABLE 5 - Growth ability of wild type and plasmid-cured HRB-1c presence and absence of heavy metals isolate in presence and absence of heavy metals Heavy metals HRB-1c Heavy metals IAA production -1 (mg ml ) Wild type Plasmid cured Untreated control 21.35 ± 0.79 Untreated control + + Cd 20.14 ± 1.60 Cd + - Co 16.97 ± 2.15 Co + - Pb 16.51 ± 3.72 Pb + - Zn 18.30 ± 1.43 Zn + - Cd, Co, Pb, Zn 14.65 p 3.27 Cd, Co, Pb, Zn + - a a Metals were supplied to LB medium as CdSO (0.25 mM), Metals were supplied to LB medium as CdSO (0.25 mM), 4 4 Co(NO ) (0.3 mM), PbCl (50 mM) and ZnSO (3 mM). Co(NO ) (0.3 mM), PbCl (50 mM) and ZnSO (3 mM). 3 2 2 4 3 2 2 4 Ann. Microbiol., 59 (1), 17-23 (2009) 21 available, a discussion on the possible differences between GM M 1 2 and untransformed trees, in terms of leaf-associated microorgan- isms would be premature. However, it is worth noting that the kb LAB isolates associated with the GM lines 5EAC1 and 12EAC1 revealed a significant degree of resistance to oxidative stress 21.2 conditions when exposed to hydrogen peroxide and UV-C radia- tion, respectively, compared to isolates derived from untrans- 5.0 formed leaves (Balestrazzi et al., unpublished results). It has been hypothesized that endophytic microorganisms 2.0 harbouring traits for the effective removal of pollutants can sig- nificantly help metal mobilization through the plant vascular sys- tem. This might be particularly relevant in poplar trees, where a prolonged time might be required in order to move contaminants 0.5 from roots to leaves. Notwithstanding the several studies carried out on soil bacteria-assisted phytoremediation (Zaidi et al., 2006; Dell’Amico et al., 2008), to date there is limited information on the potential of endophytic bacteria on the phytoremediation of heavy metal-contaminated soils. It has been suggested that the use of LB media supplement- FIG. 1 - Plasmid-profile of HRB-1c isolate. Line 1: wild type ed with heavy metals could not represent the optimal choice to cells, line 2: plasmid-cured cells, line M: Lambda DNA/ investigate the bacterial response, due to the possible chelation/ EcoRI + HindIII Marker 3 (M-Medical s.r.l.). precipitation reactions occurring in micronutrient-rich culture media. This approach has been however reported by different authors in the case of cadmium, lead, zinc and copper (Yoshida et DISCUSSION al., 1998; Shakoori and Muneer, 2002; Zhigang et al., 2006). Since the HRB-1c isolate represents the most intriguing prod- Aim of the present work was to fulfil the phenotypic characteriza- uct so far obtained from our investigation, further analyses were tion of two different collections of bacterial isolates deriving from carried out to gain information on the molecular determinants a loamy sand cultivated with transgenic white poplars expressing of its tetra-resistant phenotype. A single extrachromosomal the bar gene for herbicide tolerance (Confalonieri et al., 2000). A element was evidenced by conventional gel electrophoresis in third collection, including the epiphytic and endophytic bacteria the HRB-1c cells. The plasmid (19.0 kb in size) was removed isolated from the leaves of transgenic white poplars expressing by heat-mediated curing and resistance to Cd, Co, Pb and Zn the StSy gene for resveratrol production (Giorcelli et al., 2004; disappeared. The occurrence of larger plasmids, visible only Balestrazzi et al., unpublished results) was also examined. using more sophisticated electrophoretic approaches, cannot All the heavy metals investigated in the present work cause be excluded. In a recent work, Pereira et al. (2006) reported severe environmental pollution (Lone et al., 2008). When the that cadmium resistance was linked to high molecular weight HRB collection was analysed, resistance to several metal combi- plasmids while the czc cation efflux system, able to actively nations was evidenced. The widest range of resistance included extrude cadmium, zinc and cobalt from the cell is encoded by a four heavy metals (Cd, Co, Pb and Zn) and it was found also large plasmid (Nies, 1992). Moreover, plasmids which harbour in the HRB-1c isolate, belonging to the Bacillus genus. HRB- genes involved in biotransformation of lead have been detected 1c is characterized by traits of agronomical relevance, such as in Staphylococcus aureus and Alcaligenes eutrophus (Mergeay, a remarkable swarming ability, the consistent production of 1991). indoleacetic acid and the ability to stimulate the in vitro growth When considering the total number of isolates examined in of white poplar explants (Balestrazzi et al., 2008). It has been this study, resistance to cobalt showed the highest frequency, reported that Cd can suppress auxin production of plant-growth- since it was recovered in 94.59% of isolates. This trait is generally stimulating bacteria (Kamnev et al., 2005). The finding that the dependent on cobalt-transporting CPx-Type ATPases (Rutherford HRB-1c isolate could maintain IAA biosynthesis also in the pres- et al., 1995) and on cobalt efflux proteins (Thorgensen and ence of Cd and other heavy metals enhances the biotechnological Downs, 2007). Lead resistance occurred in 62.16% of the total value of this microorganism as a tool for bacteria-assisted phy- bacteria examined. It has been reported that, in some cases, toremediation. resistance to lead may be connected with production of extracel- Resistance to Co was a common feature of NPB isolates and lular polymers or with the intracellular sequestration of this metal it associated with resistance to Pb, Cu or Zn, thus producing by bacterial cells (Roane, 1999). different double-resistant phenotypes in isolates classified as In contrast, resistance to Cd was found only in 35.13% of Bacillus, Brevibacillus, Microbacterium and Pseudomonas. Tri- the isolates. Exposure to cadmium induces synthesis of proteins resistance, involving Co, Pb and Zn and also Cd, Co and Pb, was that export or chelate the heavy metal while the intracellular glu- detected in NPB isolates belonging to the genera Bacillus and tathione-mediated detoxification of Cd has been also described in Stenotrophomonas (Balestrazzi et al., 2007). the genus Rhizobium (Figueira et al., 2005). Finally, resistance to Multiple metal-resistance occurred in both endophytic and zinc and copper occurred with a frequency of 43.24 and 18.91%, epiphytic bacteria. Tri-resistance was observed for all LAB iso- respectively. lates deriving from the leaves of untransformed white poplar and Detailed molecular analyses of the HRB, NPB and LAB isolates in the case of the EN-12-2 associated with the GM line 12EAC1. will be required to better understand the cellular and molecular As for the GM line 5EAC1, all the tested isolates, epiphytic and mechanisms responsible for the variegated patterns of multiple endophytic, showed a double-resistant phenotype. Based on the metal-resistance so far described. Additional studies will help reported data and considering the limited number of LAB isolates distinguishing between the resistance mechanisms which rely 22 A. BALESTRAZZI et al. on chromosomal genes, plasmids or transposons and the ubiq- The investigation focused on the capacity of HRB, NPB and uitary metal tolerance ascribed to general metabolic responses. LAB isolates to withstand significant heavy metal concentra- Mechanisms responsible for metal resistance might include tions. This ability would suggest for potential applications of such exclusion by permeability barrier, intra- and extracellular seques- bacteria in the field of in situ bioremediation of polluted environ- tration, efflux pumps, enzymatic detoxification, proteome and ments. Specific isolates with multiple metal-resistance might be metabolome changes (Silver, 1992; Nies, 1999; Baker-Austin et useful to target different elemental pollutants at contaminated sites. Furthermore, the ability to interact with perennial trees, al., 2006). Several studies have documented the predominance of e.g. those included in the genus Populus, might help to obtain Gram-negative bacteria in metal-contaminated soils and this novel plant-microbe remediation systems. finding might be explained by the fact that the Gram-negative Acknowledgments cell wall is a more efficient barrier to toxic metals than the This research was supported by a grant from Regione Lombardia Gram-positive one (Duxbury and Bicknell, 1983). In accord- (Divisione Generale Agricoltura). M.B. received a doctoral fellow- ance with this hypothesis, although most of the environmental ship from Regione Lombardia. isolates (62.1%) examined in this study were Gram-positive, the tetra-resistant phenotypes were more frequent among the Gram-negative bacteria belonging to the genus Pseudomonas. REFERENCES The wide range of heavy metal resistant phenotypes rou- tinely isolated from soil bacteria might be related to the common Baker-Austin C., Wright M.S., Stepanauskas R., McArthur J.V. agricultural practices which rely upon the use of metal-containing (2006). Co-selection of antibiotic and metal resistance. products, such as fertilizers, fungicides, pesticides and her- Trends Microbiol., 14: 176-182. bicides, routinely adopted to enhance crop production (He et al., 2005). It is worth noting that both the herbicide-tolerant Balestrazzi A., Allegro G., Confalonieri M. (2006). Genetically and resveratrol-producing white poplars grown in greenhouses modified trees expressing genes for insect pest resistance. underwent intensive fertilization treatments and nutrients solu- In: Fladung M., Ewald D., Eds, Tree Transgenesis: Recent tions enriched in microelements were supplied to support growth Developments, Springer-Verlag Berlin Heidelberg, pp. 253- (Balestrazzi et al., 2008). The heavy metals tested in this work 273. include micronutrients such as Co, Cu and Zn which are essen- Balestrazzi A., Bonadei M., Carbonera D. (2007). Nuclease- tial components of redox processes and cofactors of relevant producing bacteria in soil cultivated with herbicide resistant enzymes (O’Halloran, 1993). It is known that bacteria possess transgenic white poplars. Ann. Microbiol., 57: 531- 536. copper resistance operons which display metal-responsive tran- Balestrazzi A., Bonadei M., Zelasco S., Quattrini E., Calvio C., scriptional regulation with membrane-spanning proteins involved Galizzi A., Carbonera D. (2008). Recovery of useful traits in monitoring changes in copper concentrations in periplasm from isolates inhabiting an agricultural soil cultivated with (O’Halloran, 1993). On the other hand, zinc is found in enzymes herbicide-resistant poplars. Can. J. Microbiol., 54: 201-208. involved in transcription and in transcription factors respon- Castiglione S., Franchin C., Fossati T., Lingua G., Torrigiani P., sive to a variety of intra- and inter-cellular signals (O’Halloran, Biondi S. (2007). High zinc concentrations reduce rooting 1993). Cadmium is the only non essential element evaluated in capacity and alter metallothionein gene expression in white the present work. Resistance to essential elements is usually poplar (Populus alba cv. Villafranca). Chemosphere, 67: chromosome-based while the resistance to non essential trace 1117-1126. elements is associated with plasmids and transposons (Silver, Collins Y.E., Stotzky G. (1989). Factors affecting the toxicity of 1992). This is in accordance with the reported data linking the heavy metals to microbes. In: Beveridge T.J., Doyle R.J., Eds, resistance to cadmium and the 19-kb plasmid detected in HRB-1c cells. Within this context the correlation between this extracel- Metal Ions and Bacteria, John Wiley and Sons Inc., New York, pp. 31-90. lular element and resistance to Co, Pb and Zn remains difficult to explain. However, in case of plasmid-determined copper resist- Confalonieri M., Belenghi B., Balestrazzi A., Negri S., Facciotto G., ance mechanisms involving bioaccumulation, sequestration and Schenone G., DelleDonne M. (2000). Transformation of elite efflux have been documented (Silver, 1992). white poplar (P. alba) cv Villafranca and evaluation of herbi- Finally, it has been reported that heavy metal toxicity might cide resistance. Plant Cell Rep., 19: 978-982. be lowered in soils with high clay, since the charged clay parti- Dell’Amico E., Mazzocchi M., Cavalca L., Allievi L., Andreoni V. cles limit metal availability by sequestration (Collins and Stotzky, (2008). Assessment of bacterial community structure in 1989). The medium-textured loamy sand utilized for white poplar a long-term copper-polluted ex-vineyard soil. Microbiol. cultivation in greenhouse was characterized by a reduced clay Res.,163: 671-683. content (5.62%) (Balestrazzi et al., 2007, 2008) and this might Diaz-Ravina M., Baath E., Frostegard A. (1994). Multiple heavy have contributed to the high frequency of multiple metal-resist- metal tolerance of soil bacterial communities and its meas- ance detected in the bacterial isolates. urement by a thymidine incorporation technique. Appl. The response of HRB, NPB and LAB isolates to a wider range Environ. Microbiol., 60: 2238-2247. of trace elements, including As, Se and Hg, will possibly reveal Duxbury T., Bicknell B. (1983). Metal-tolerant bacterial popu- additional resistance traits useful for waste bioremediation or lations from natural and metal polluted soils. Soil Biol. biometallurgy. To date, microbial studies are required to identify Biochem., 9: 105-112. novel soil culturable microorganisms able to support the phy- toremediation performace of specific plant systems (Lone et al., Figueira E.M.A.P., Lima A.I.G., Pereira S.I.A. (2005). Cadmium 2008). The present work presents a list of novel environmental tolerance plasticity in Rhizobium leguminosarum bv. viciae: isolates with multiple metal-resistance which will be useful in glutathione as a detoxifying agent. Can. J. Microbiol., 51: future studies on bacteria-assisted phytoremediation with trees. 7-14. Ann. Microbiol., 59 (1), 17-23 (2009) 23 Giorcelli A., Sparvoli F., Mattivi F., Balestrazzi A., Tava A., Ewald D., Eds, Tree Transgenesis: Recent Developments, Vrhovsek U., Bollini R., Confalonieri M. (2004). Expression of Springer-Verlag Berlin Heidelberg, pp. 253-273. stilbene synthase (StSy) gene from grapevine in transgenic Roane T.M. (1999). Lead resistance in two isolates from heavy white poplar results in high accumulation of the antioxidant metal-contaminated soils. Microb. Ecol., 37: 218-224. compounds resveratrol glucosides. Trans. Res., 13: 203- Rutherford J.C., Cavet J.S., Robinson N.J. (1995). Cobalt- dependent transcriptional switching by a dual-effector MerR- Gonzalez J.M.Jr, Carlton B.C. (1984). A large transmissable plas- like protein regulates a cobalt-exporting variant CPx-type mid is required for crystal toxin production in Bacillus thur- ATPase. J. Biol. Chem., 274: 25827-25832. ingiensis variety israelensis. Plasmid, 11: 28-38. Ryan R., Ryan D., Dowling D. (2008). Multiple metal resistant Gordon S.A., Weber R.P. (1951). Colorimetric estimation of transferable phenotypes in bacteria as indicators of soil con- indoleacetic acid. Plant Physiol., 26: 192-195. tamination with heavy metals. J. Soils Sedim., 5: 95-100. He Z.L., Yang X.E., Stoffella P.J. (2005). Trace elements in agr- Sambrook J., Fritsch E.F., Maniatis T. (1989). Molecular Cloning: A nd oecosystems and impacts on the environment. J. Trace Elem. Laboratory Manual, 2 edn., Cold Spring Harbor Laboratory Med. Biol., 19: 125-140. Press, Cold Spring Harbor, New York. Jing Y., He Z., Yang X. (2007). Role of soil rhizobacteria in phy- Shakoori A.R., Muneer B. (2002). Copper-resistant bacteria from toremediation of heavy metal contaminated soils. J. Zhejiang industrial effluents and their role in remediation of heavy Univ. Sci. B, 8: 192-207. metals in wastewater. Folia Microbiol., 47: 43-50. Kamnev A.A., Tugarova A.V., Antonyuk L.P., Tarantilis P.A., Siciliano S.D., Fortin N., Mihoc A., Wisse G., Labelle S., Beaumier Polissiou M.G., Gardiner P.H. (2005). Effects of heavy metals D., Ouellette D., Real R., Whyte L.G., Banks M.K., Schwab P., on plant-associated rhizobacteria: comparison of endophytic Lee K., Greer C.W. (2001). Selection of specific endophytic and non-endophytic strains of Azospirillum brasilense. J. bacterial genotypes by plants in response to soil contamina- Trace Elem. Med. Biol., 19: 91-95. tion. Appl. Environ. Microbiol., 67: 2496-2475. Kuffner M., Poschenrieder M., Wieshammer G., Gorfer M., Silver S. (1992). Plasmid-determined metal resistance mecha- Sessitsch A. (2008). Rhizosphere bacteria affect growth and nisms: range and overview. Plasmid, 27: 1-3. metal uptake of heavy metal accumulating willows. Plant Thorgensen M.P., Downs D. (2007). Cobalt targets multiple Soil, 304: 35-44. metabolic processes in Salmonella enterica. J. Bacteriol., Lingua G., Franchin C., Todeschini V., Castiglione S., Biondi S., 189: 7774-7781. Burlando B., Parravicini V., Torrigiani P., Berta G. (2008). Yoshida N., Murooka Y., Ogawa K. (1998). Heavy metal particle Arbuscolar mycorrhizal fungi differentially affect the response resistance in Thiobacillus intermedius 13-1 isolated from cor- to high zinc concentrations of two registered poplar clones, roded concrete. J. Ferment. Bioeng., 85: 630-633. Villafranca (Populus alba L.) and Jean Pourtet (Populus nigra Zaidi S., Usmani S., Singh B.R., Musarrat J. (2006). Significance L.). Environ. Pollut., 153: 137-147. of Bacillus subtilis strain SJ-101 as a bioinoculant for con- Lone M.I., He Z., Stoffella P.J., Yang X. (2008). Phytoremediation current plant growth promotion and nickel accumulation in of heavy metal polluted soils and water: progresses and per- Brassica juncea. Chemosphere, 64: 991-997. spectives. J. Zhejiang Univ. Sci. B, 9: 210-220. Zelasco S., Reggi S., Calligari P., Balestrazzi A., Bongiorni C., Mendez M.O., Maier R.M. (2008). Phytostabilization of mine tail- Quattrini E., Delia G., Bisoffi S., Fogher C., Confalonieri M. ings in arid and semiarid environments - an emerging reme- (2006). Expression of the Vitreoscilla hemoglobin (VHb)- diation technology. Environ. Health Perspect., 116: 278-283. encoding gene in transgenic white poplar: plant growth and biomass production, biochemical characterization and cell Mergeay M. (1991). Towards an understanding of the genetics of survival under submergence, oxidative and nitrosative stress bacterial metal resistance. Trends Biotechnol., 9: 17-24. conditions. Mol. Breed., 17: 201-216. Nies D.H. (1992). Resistance to cadmium, cobalt, zinc and nickel Zelasco S., Ressegotti V., Confalonieri M., Carbonera D., Calligari in microbes. Plasmid, 27: 17-28. P., Bonadei M., Bisoffi S., Yamada K., Balestrazzi A. (2007). Nies D.H. (1999). Microbial heavy metal resistance. Appl. Evaluation of MAT-vector system in white poplar (Populus Microbiol. Biotechnol., 51: 730-750. alba L.) and production of ipt marker-free transgenic plants O’Halloran T. (1993). Transition metals in control of gene expres- by ‘single-step transformation’. Plant Cell Tiss. Org. Cult., sion. Science, 261: 717-725. 91: 61-72. Pereira S.I., Lima A.I., Figueira E.M. (2006) Screening possible Zhigang A., Cuijie L., Yuangang Z., Yeljie D., Watcher A., Gromes mechanisms mediating cadmium resistance in Rhizobium R., Rausch T. (2006). Expression of BjMT2, a metallothionein leguminosarum bv. viciae isolated from contaminated portu- 2 from Brassica juncea, incresaes copper and cadmium toler- guese soils. Microb. Ecol., 52: 176-186. ance in Escherichia coli and Arabidopsis thaliana, but inhibits Peuke A.D., Rennenberg H. (2006). Heavy metal resistance and root elongation in Arabidopsis thaliana seedlings. J. Exp. Bot., phytoremediation with transgenic trees. In: Fladung M., 57: 3575-3582.

Journal

Annals of MicrobiologySpringer Journals

Published: Nov 24, 2009

There are no references for this article.