DETECTION OF GENES RESPONSIBLE FOR HEAVY METALS RESISTANCE IN LOCALLY ISOLATED PSEUDOMONAS SPP.

Plant growth-promoting rhizobacteria (PGPR) that can tolerate heavy metals, provide the basis for microbial inoculums showing heavy metals tolerance properties. This study was aimed to detect the heavy metal resistance genes in plant-growth-promoting Pseudomonas spp. isolated from many agricultural fields. The collected isolates were screened for their plant growth-promoting (PGP) traits, hydrolytic enzymes, Siderophore, ammonia, and indole-3-acetic acid (IAA). Then, subjected to concentrations of CuSO 4 , CdCl 2 , and ZnCl 2 to determine the minimum inhibitory concentration (MIC). The DNA was extracted from the selected isolates then PCR test was achieved to detect copA , copB , and czcA genes, responsible for heavy metal resistance. Seventy Pseudomonas spp. isolates were obtained; 41 (58.57%), 6 (8.57%), and 15 (21.42%) isolate produced protease, cellulase, and pectinase, respectively. The isolates were positive for siderophore and ammonia production. However, 68 (97.14%) isolates have produced indole-3-acetic acid. Eight isolates were selected and identified as Pseudomonas aeruginosa using the Vitek 2 compact system. The isolates' resistance to heavy metals differed significantly. The isolate B49 had a higher resistance to CuSO 4 (MIC = 3200 µg/ml) and ZnCl 2 (MIC = 2600 µg/ml), while the isolate B66 recorded a higher resistance to CdCl 2 (MIC = 1000 µg/ml). copB , and czcA genes were detected in the eight P. aeruginosa isolates, while copA gene was detected in seven, except B69.


INTRODUCTION
Microorganisms in the rhizospheric soil range from beneficial to pathogens.The beneficial bacteria such as Pseudomonas spp.and Bacillus spp.have shown a potential role in improving plant growth through nutrient provide and producing many biological control agents to face the disease-causing microorganisms (10,42).Pseudomonas species represent essential PGPR (Plant growth-promoting rhizobacteria) that increase crop yield through direct and indirect methods.PGPR have several processes for controlling plant pathogens and they also compete with phytopathogens for resources and space (14,38).Pseudomonas spp. is ubiquitous bacteria with various applications because they exhibit a many of diverse traits in different environments, was utilized as a biocontrol agent, plant growth promoter, source of antibiotics and efficient bioremediation strains.Plant growth-promoting systems traditionally grouped direct and indirect processes (13).Heavy metals, such as Zn, Cd, Ni, Cu, Pb, Cr and Hg, are a common problem in agricultural soil fields, and it is frequently caused by industrial processes located nearby (9,20).These metals are hard to remove from the environment, differ from many pollutants that can be decomposed biologically or chemically and are ultimately undegradable, so their harmful effects last longer (3, 6).Bacteria have evolved numerous resistance mechanisms to cope with heavy metal stress.Several resistance mechanisms involve heavy metal, complexation, sequestration, metal conversion to a less hazardous species, and direct metal efflux out of the cell.Metals crossing the bacterial cell may interact in many ways based on their chemical properties and concentration.When exposed to high levels, cells usually respond by expressing unique systems of resistance (P-type ATPases, CDF (cation diffusion facilitator transporters), resistancenodulation-cell division) RND(efflux pumps, metallothioneins)(35).Heavy metal resistance responsible genes copA, copB, and czcA were found in gram-negative bacteria such as Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Acinetobacter baumannii, and Xanthomonas spp.CopA is a P-type ATPase found in the periplasmic membrane, and CopB is a copper-binding protein in the outer membrane that protects against copper.The czcA gene is a member of the Resistance Nodulation-Division (RND) family found in the periplasm that serves as a metal efflux pump protein for Cd and Zn (16,23,29).The objective of the current study was to evaluate Pseudomonas spp.isolates as plant growth promoters and heavy metals resistant and detect the presence of genes responsible for their resistance to Cu, Cd, and Zn.Serial dilutions were prepared from different plants' rhizosphere. 1 g of soil was added to a test tube was contained 9 ml of H 2 O (0.85% NaCl), mixed thoroughly, and left to stand before they formed successive serial dilutions.A 100 µl of each 10 -4 , 10 -5 , and 10 -6 dilution was spread on the surface of three agar media: King's B, Pseudomonas isolation agar (PIA), and Cetrimide agar, then incubated at 28°C for 48 h and re-cultured on the same media.Different colonies were purified in MacConkey agar medium and subjected to biochemical tests.
Clear yellow zones development indicates a positive result for cellulase production (19).Pectinase production was tested by combining pectin with M9 medium, 1 g (NH 4 ) 2 SO 4 , 1 g Dextrose, 10 g pectin, 0.5 g sodium citrate, 1.2 g yeast extract, 20 g agar (g L -1 ).The formation of a clear zone when plates flooded with Lugol's iodine solution for 10 minutes after three days of incubation at 28°C denotes the isolate's ability to produce pectinase (32).Ammonia and siderophore production The bacterial isolates were inoculated in 10 ml peptone water for ammonia synthesis.After 48-72 h of incubation at 28 °C, the culture was centrifuged, and the supernatant loaded with 1 ml of freshly prepared Nessler's reagent, the positive test results a brown-yellow colour (34).Siderophore production was determined on a nutrient agar (NA) supplemented with chelating 2,2-Bipyridyl solution (2 mg L -1 ), which filtered in NA at 45°C The growth of isolates signalled a positive test (31).Indole acetic acid (IAA) production IAA production was determined using Erlenmeyer flasks containing 50 ml of Nutrient broth medium (NB) with 0.2% (v/v) L-tryptophan.Flasks were inoculated with 2% of bacterial growth (0.5 O. D; 1 × 10 8 CFU mlˉ¹) and subjected to 120 rpm in a shaker incubator at 28°C for 48 h. 10 ml of growth sample was pipetted from each flask and centrifuged for 15 min at 10000 rpm.The supernatant was mixed with twice the volume of Salkowski'sr reagent (1 ml of 0.5M FeCl 3 and 49 ml of 35% HCIO 4 ) and re-incubated for 30 min at 25°C in a dark place, then estimated calorimetrically at 530 nm (12).

Identification of bacterial isolates
Bacterial isolates were identified using the Vitek 2 compact system.The activated isolates, on MacConkey ager at 28°C for 24 hours, were used to prepare bacterial suspension by transferring to 3 ml of sterile saline.The turbidity was fixed at 0.5 OD using the McFarland standard.The kit was added for each tube and incubated for 18 hours in the device.

MIC determination
The bacterial isolates were subjected to CuSO 4 , CdCl 2 , and ZnCl 2 on Mueller-Hinton agar plate by a gradient concentration of heavy metals.The concentration level of heavy metals was started at 100 μg/ml and increased by 50 μg/ml, except for the HgCl 2 increased by 10 μg/ml, plates then incubated for 48 h at 28°C.MIC was determined on the plate medium when there was no visible growth (1) DNA Extraction DNA from the selected isolates were extracted using the ABIOpureTM Total DNA kit (ABIOpure, USA), according to the company protocol.A Quantus Fluorometer was used to quantify the nucleic acid concentration to detect the integrity of DNA for downstream applications.For DNA measurement, one μl of DNA was added to 199 μl of diluted QuantyFlour Dye and incubated at room temperature for five minutes.Detection of heavy metal resistance genes PCR test was used to detect heavy metal resistance genes using specific primers (Table 1) for amplifying the region 475bp 364bp and 206bp of copA, copB, and czcA, respectively.The PCR reaction was carried out in a 20μl volume, which was composed of 10μl master mix, 1μl forward primer, 1μl reverse primer, 2μl DNA template, and 6μl of nuclease-free water.The program used was illustrated in the Table 2.The PCR products were analysed on the agarose gel electrophoresis (1.5%, w/v), formed from 1X TAE buffer with 1μl of ethidium bromide (10mg/ml).The wells were loaded with PCR products (10μl), then powered at 100v/mAmp for 75 minutes.After that, the stained bands were displayed under the UV Transilluminator.

Statistical analysis
The data were statistically analyzed using ANOVA by the Statistical Analysis System program to compare the means of triplicate samples with the least significant difference (LSD) values (36).

RESULTS AND DISCUSSION
Isolation of Pseudomonas spp.Bacterial isolates were gained from different rhizospheric soils, and characterized depending on morphological, cultural, and biochemical characteristics.The isolates were positive for oxidase, catalase tests, Simon citrate, and motile, while negative for Gram stain, indole, Voges-Proskauer, methyl red and starch hydrolysis.Accordingly, seventy isolates were mostly referred to Pseudomonas spp.(15).
Lytic enzymes can inhibit the growth or suppression of phytopathogens by degrading the fungal cell walls.Chitin and fibrils of glucan are incorporated into the protein matrix, that's why proteases play an essential role in fungus cell wall destruction (22).Alsalim (8) reported the production of hydrolytic enzymes by Pseudomonas spp.and found that all isolates were positive for protease and pectinase, while 66.6% of them could produce cellulase.Other researchers isolated 87 Pseudomonas species from the tomato (Lycopersicon esculentum) rhizosphere.They reported that only 30, 28, and 12 isolates were positive for protease, cellulase, and pectinase, respectively.Also found that the lytic enzymes in 30 isolates have antagonistic activity against the plant pathogenic bacterium Ralstonia solanacearum (39).Another study isolated three Pseudomonas fluorescens isolates from the plant rhizosphere, their results showed that the three strains were positive for protease, while negative for cellulase, chitinase, and glucanase (41) Siderophore and ammonia production In this study, the siderophore test was positive for all the isolates (Table 3).The seventy isolates revealed a growth on the 2,2 dipyridyl-containing agar media after 48 hours of incubation, which could contribute to their antifungal activity.The qualitative test of ammonia production in peptone water revealed that all isolates were positive, the colour changed to yellow or brown in peptone water of old culture.In their study, Kotasthane et al. (24) mentioned that out of twenty-nine only eight isolates produced siderophores in the presence of 8-Hydroxyquinoline (50 mg/l).The eight isolates have antagonistic effects against Rhizoctonia solani and Sclerotium rolfsii.In another study, P. asplenii, P. fluorescens, and P. aeruginosa isolates showed a high level of inhibition against Rhizoctonia solani, 93.15%, 88.70%, and 86.85%, respectively, with the ability to synthesise siderophore and ammonia production ability of all the isolates (5).Agrawal et al. (2) reported that twentyfour fluorescent Pseudomonas isolates subjected to different siderophore assays were shown to produce siderophores on an irondeficient succinate medium.Rana et al. (34) reported that twenty Pseudomonas fluorescent isolates, collected from apple orchards, were exhibited a positive result in ammonia and siderophore synthesis.

Indole-3-acetic acid (IAA) production
The amount of indole-3-acetic acid produced by Pseudomonas spp.isolates, evaluated quantitively in NB broth, was ranged from 1.20±0.03 to 35.15±0.76μg/ml (Table 3).IAA has a role in the extension, division, and differentiation of plant cells.IAA also promotes the growth of lateral roots and root hairs, which increases nutrient uptake by surfaces and may result in higher levels of nutrient absorption and dramatically improve the plant's shoot length (40).The highest IAA producing strain was B49 (35.15±0.76μg/ml) followed by B46 (28.59±0.05μg/ml) and B4 (26.98±0.42μg/ml).Sandilya et al. (37) reported that eight Pseudomonas spp.isolates derived from castor rhizosphere showed IAA production in vitro ranged from 5.19 μg/ml to 27.84 μg/ml.The strain RTE4 of Pseudomonas aeruginosa, isolated from tea rhizosphere, was tested by Chopra et al. (13), and they found high indole acetic acid production (74.54 µg/ml) after seven days of incubation.Wadekar and Kagne (40) isolated 25 Pseudomonas spp.from the rhizosphere of soybean.Their results showed the highest IAA production, which reached 19.34 µg/ml, was by the strain S3 after 48 h of incubation on King's B. Akter et al. (5) reported that P. asplenii and P. fluorescens isolates collected from rice plants were shown to have a maximum IAA production reached 51 µg/ml and 52 µg/ml, respectively, while P. aeruginosa did not produce IAA.Ali et al. (7) found maximum IAA production by P. aeruginosa (8.95 µg/ml) during testing 12 bacterial isolates.Two isolates belonging to P. aeruginosa (JB and JC) were isolated from Solanum melongena and Capsicum annuum and synthesised IAA by 15.54 and 23.38 µg/ml, respectively (30).

Isolates identification
Based on the previous data, eight isolates (Table 3) were selected and identified by the Vitek 2 compact system.The results revealed that all belong to Pseudomonas aeruginosa.

Heavy metals resistance
The resistance profile to the heavy metals CuSO 4 , CdCl 2 , and ZnCl 2 for eight P. aeruginosa isolates, was carried out on Mueller-Hinton agar.The results in Table 4 demonstrate that MIC values to the Copper were extending from 800-3200 μg/ml.The B49 strain has recorded a higher MIC value (3200 μg/ml) which differs significantly from other strains, followed by the B46 strain (2800 μg/ml).The MIC values for Cadmium resistance were ranging from 600-1000 μg/ml and the strain B66 recorded a higher MIC (1000 μg/ml) differs significantly from other strains.The resistance to Zinc, represented in the form of MIC values, was extending from1050-2600 μg/ml.The strain B46 recorded the value 2600 μg/ml which differs significantly from the other strains.Metals resistances were appeared to be heterogeneous among P. aeruginosa isolates, and B49 has a higher resistance to copper sulphate and zinc chloride when compared to other bacterial isolates Ghaima et al. (17).found that the MIC rates of cadmium have been ranging from 600 to 900 mg/l for P. aeruginosa that isolated from Al-Dora agricultural soil, while the MIC for Copper and Cadmium was 3600 μg/ml for the strain KZ5 of Pseudomonas aeruginosa, according to Benhalima et al. (11).Akinbowale et al. (4) isolated 129 strains of Pseudomonas from different Australian farms.The majority of the isolates showed 800 μg/ml MIC values for Cd and Co, more values for Cu and Pb (1600 μg/ml), and the highest for Cr and Pb (3200 μg/ml), while MIC for Mn was more than 3200 µg/ml.Pseudomonas aeruginosa exhibited a resistance to Pb 2+ Cu 2+ , Cd 2 , the minimum inhibitory concentration ranged between 100 and 500 ppm, while it was showed no resistance, no growth, in the presence of Ag 2+ and Hg 2+ (28).Malik and Aleem (26) isolated 96 strains of Pseudomonas spp.from soils, they noticed that most isolates had MIC values up to 3,200 μg/ml and some up to 1,600 μg/ml, and 91.6% of them were resistant to Pb 2+ and Cu 2+ , while 62.5% were resistant to Zn 2+ .Another study by Imron et al. (21) mentioned that P. aeruginosa isolated from sanitary landfills exhibited MIC values of more than 20 mg l − 1 for Zn, Mg Cd and Pb, using the disk diffusion method.

Pseudomonas aeruginosa
The eight studied P. aeruginosa isolates were subjected to PCR amplification to detect the presence of the genes responsible for their resistance to Cu, Cd, and Zn, copA, and copB genes for Cu and czcA gene for Cd, and Zn.The copB and czcA genes were observed in all eight isolates, while the copA gene was observed in only seven isolates (Figure 1).The copA and copB genes' presence were confirmed by many researchers in P.
aeruginosa that showed heavy metal resistance.Martins et al. (27) isolated two tetracycline-resistant strains of P. aeruginosa (EW32 and EW33) from a polluted environment and detected copA, copB and czcA genes presence using PCR.They said that treatment of numerous pathogenic bacteria in humans and animals can be limited due to the exchange of resistance genes between environmental and pathogenic bacteria.Their study observed that the EW32 strain was positive for copA, copB and czcA genes, while the EW33 strain was positive only for the czcA gene.Pitondo-Silva et al. (33) detected the occurrence of the genes in sixty-four P.
aeruginosa that were isolated from different crops of five Brazilian regions.Their study showed that the most common existence of heavy metals resistance genes for copB, copA and czcA were 65%, 48% and 46%, respectively.Li et al. (25) mentioned that copA and lipoprotein had a role in the sequestration and efflux of copper out of the cytoplasm and proposed a response model for Pseudomonas spp.under higher concentrations of copper.They noted the reduction in cell size, which leads to reducing the quantity of copper bonded around the cell surface, and less energy had required to maintain themselves during copper stress.