Uranium (VI) Complexation with Siderophores-pyoverdine Produced by Pseudomonas Fluorescens SHA 281

Inte rnational Journal of Nucle ar Ene rgy Science and Engine ering Volume 3 Issue 4, De cember 2013 doi: 10.14355/ijnese.2013.0304.03

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Uranium (VI) Complexation with Siderophores-pyoverdine Produced by Pseudomonas Fluorescens SHA 281 Shimaa S. Hussien1 , Osman A. Desouky2 , Mahmoud E.F. Abdel-Haliem 3 , Abdou A. El-Mougith4 Nuclear Materials Authority, P.O. Box 530 El-Maadi, Cairo, Egypt

1,2

Department of Botany, Faculty of Science, Zagazig University, Egypt

3,4

College of Science and Arts at Nayriya, Dammam University, Kingdom of Saudia Arabia

3

mash_1997@yahoo.com

1

Abstract Fluore sce nt Pseudomonas species produce pyove rdine -type siderophores with a high pote ntial to dissolve ; bind and transport uranium in the e nvironment. Ide ntification of Pseudomonas fluorescens SHA 281 was confirme d by 16S rDNA se que ncing. The pyoverdine produce d by the tested strain was e xtracte d and purifie d using XAD-4 Ambe rlite re sin and its chemical structure was confirme d using NMR and CA-MS analyses. The ge l e lectrophoresis of pyoverdine appeare d low molecular we ight with one prote in band at ~12.5 kDa. The ge ne responsible for pyoverdine production was ide ntifie d using PCR technique . Unde r optimum conditions for comple xation, it was found that pyoverdine comple xe d with about 90.48 % and 86.41 % of U (VI) from monazite and (Th-U) conce ntrate, respectively.

Keywords Uranium; Pyoverdines; Complexation; Pseudomonas Fluorescens

Abbreviations: Common amino acids, 3-letter code; OHAsp, _β hydroxy Asp; cOHOrn, cyclo-N5-hydroxyOrn(3-amino-1-hydroxy-piperidone-2); Chr, pyoverdine chromophore; Suca, succinamide residue; MS, mass spectrum or spectrometry; CA, collision activation; NMR, Nuclear magnetic resonance; kDa, killo dalton; SDS, sodium dodecyl sulfate; Pvd, pyoverdine and PVdS, siderophores-pyoverdine gene.

Introduction Extensive knowledge of the interactions which occur between radionuclides and the biosphere is necessary in order to predict the possible migration of these elements. Pseudomonas fluorescens is one of the fluorescent pseudomonads that secrete pyoverdines for its essential requirement for iron (Meyer, 2000).

Pyoverdine is a water soluble, yellow-greenish fluorescent siderophore involved in high affinity transport of iron into the cell due to presence of a

chromophore (Budzikiewicz, 1993). Structurally, it can be divided into three different parts: (a) a peptide chain composed of 6 to 12 mainly hydrophilic amino acids bound via their N-termini to the carboxyl group of the chromophore, (b) the chromophor(1S)-5-amino-2, 3-dihydro-8, 9-dihydroxy-1H-pyrimido[1,2-a] quinoline -1-carboxylic acid, and (c) an acyl chain attached to the NH2 group of the chromophore consisting of dicarboxylic acid residues. Succinate or its amide form depends on the growth conditions of the producer microorganism (Meyer, 2000). Siderophores are relatively low-molecular-mass (<14 kDa), that are usually contain catecholate or hydroxamate functional groups (Crichton, 1991 and Drechsel et al., 1995). Siderophores and their analogues have also been investigated for possible medical use in the decorporation of actinides from the body (Stradling, 1998) while simple hydroxamates may have been used in the reprocessing of nuclear fuel (Taylor et al., 1998). Later, pyoverdinestype siderophores excreted by a groundwater strain of Pseudomonas fluorescens were found to efficiently mobilize uranium and other trace elements from shale mine tailings (Kalinowski et al., 2004).

Materials and Methods Strain Isolation and Identification 1) Tested Strain Pseudomonas fluorescens SHA 281 strain was obtained from the Laboratory of Microbiology, Botany Department, Faculty of Science, Zagazig University.

2) 16S rDNA Sequencing The identification of bacterial strain was confirmed by 16S rRNA sequencing analysis.

The universal primers (forward primer [F27]5'-

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Inte rnational Journal of Nucle ar Ene rgy Science and Engine ering Volume 3 Issue 4, De cember 2013

AGAGTTTGATCCTGGCTCAG-3' (Chénbey, et al., 2000) and reverse primer [R1492] 5'GGTTACCTTGTTACGACTT-3') (Turner, et al., 1999) were used.

Media and Growth Conditions Pseudomonas fluorescens SHA 281 was inoculated into sterile succinic acid medium (Maria & Pillar, 2002).

The standard succinic acid medium (SA) contained (g/l) K2 HPO4 , 6.0; KH2PO4, 3.0; (NH4)2SO4, 1.0; .7 H2O, 0.2; C4H4Na 2O4.6H2O, 4.0.

Nonspecific Assay for Siderophores-pyoverdine Production in Culture Supernatant 50 µl of culture supernatant was mixed with 0.12 M FeCl3 in 0.005 N HCl to give a final volume of 900 µl (Haydon et al., 1973), and the absorbance at 425 nm was determined. Detection of catechol and hydroxamate functional groups was achieved as described by (Arnow, 1937 & Emery and Neilands, 1960).

Estimation of Siderophores-pyoverdine The produced siderophore was estimated as previously mentioned by (Schwyn and Neilands, 1987).

Siderophores-pyoverdine Recovery The pyoverdine was purified from the filtered supernatant as outlined in (Meyer and Abdallah, 1978).

Characterization of Extracted Siderophores-pyoverdine 3) Proton NMR Analysis of P urified Siderophorespyoverdine Proton nuclear magnetic resonance (P-NMR) technique was done using deuterated dimethyl sulphoxide (DMSO) solvent. The siderophore sample was dissolved in 0.75 ml DMSO and injected through a 5-mm OD NMR tube to record the nuclear magnetic resonance (1 H) (NMR) at 70 eV. The (1 H) P-NMR spectra were recorded on a Shimadzu GCMS-Qp-1000EX spectrometer.

4) Analysis of the Siderophores-pyoverdine Using Mass Spectrometry Mass spectra were obtained using a Shimadzu Qp2010-plus mass spectrometer. The spray voltage was 70 KV and the capillary temperature was at 220 oC. 5) Determination the Siderophores-pyoverdine

Molecular

Weight

of

The molecular weight of the extracted siderophores in sample was determined using sodium dodecyl 96

sulphate polyacrylamide gel electrophoresis (SDSPAGE) according to Studier (1973).

6) Identification of Siderophores-pyoverdine Gene (PvdS) Total DNA was extracted from isolated colony of bacteria (Sambrook and Russel 2001). The PCR amplification was carried out in a Gen e-Amp PCR system 9600 thermocycler (Perkin Elmer). The resulting DNA sequences were compared using the Basic Local Alignment Search Tool (BLAST) program (Altschul et al., 1997).

Complexation of Uranium with Siderophores-pyoverdine This assay method was developed to screen the capability of uranium to complex with siderophorespyoverdine. Pyoverdine in supernatant, extracted by ethyl acetate and purified by XAD-4 Amberlite resin were used. The concentration of uranium (VI) was 100 ppm in all cases. 20 ml of solution contains pyoverdine mixed with 100 ppm uranium at room temperature for 20 min.

Factors Affecting Complexation of Uranium from Bioleached Liquors of Monazite and (Th-U) Concentrate with Siderophores-pyoverdine The tested ores were bioleached by P. fluorescens SHA 281 under the same conditions as previously mentioned by (Hussien, 2011). Certain factors were studied to determine the suitable conditions for maximum complexation of uranium from the bioleached liquors of monazite and (Th-U) concentrate with pyoverdine. Different pyoverdine concentrations (25, 50, 90, 120 and 150 mg/20 ml); pH values (2.5, 4.5, 6.5, 8.5 and 10.5); shaking times (10, 20, 30, 40, and 50 min) and shaking speeds (100, 150, 175, 200 and 250 rpm) were tested.

Results Strain Identification The bacterial strain was confirmed by 16S rDNA sequence and the results indicated that, there was more than 99% identity in nucleotide level closely related to Pseudomonas fluorescens. The resulting DNA sequences of tested bacteria by F27 primer were deposited to the GenBank with The accession number BankIt1555892 JX455162.

Nonspecific Assay for Siderophores-pyoverdine in Culture Supernatant Supernatant of P. fluorescence SHA 281 produced a

Inte rnational Journal of Nucle ar Ene rgy Science and Engine ering Volume 3 Issue 4, De cember 2013

stable yellow color measurable at 425 nm when mixed with FeCl3 or Fe(ClO4 ), indicating the presence of siderophores-pyoverdine. Slope values for both nonspecific siderophore produced in king′s A (KA) (Holt et al., 1994) and SA media assays are tabulated in Table (1) and also, detection of catechol and hydroxamate functional groups was also detected in

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the same table.

Estimation of Siderophores-pyoverdine The pyoverdine yield was estimated by solvent extraction using ethyl acetate and crude pyoverdine production was reported to be 59 mg/100 ml of succinic acid medium.

TABLE (1) NON SPECIFIC AND SPECIFIC ASSAYS FOR SIDEROPHORES -PYOVERDINE PRODUCED BY

Specific Catecholb FeCl3 425 nm Color in acid Color in base King′s A medium (KA) 1.599 Yellow None S uc c inic acid medium (SA) 1.792 Yellow None

P. fluorescens S HA 281

Nonspecifica

Growth medium

Hydroxamate c Color 264 nm None 2.341 None 2.771

a. Slope of iron-binding ac tivity equals the absorbance at 425 nm×10 3 divided by percent c ulture supernatant. b. Catec hol derivatives are detec ted by the formation of a yellow color at ac id condition and a red color at basic conditions. c. Hydroxamate derivatives are detec ted by the formation of nitrosodimer whic h absorb specially at 264 nm.

FIG. (1) 1 H NMR S PECTRA OF PYOVERDINE-S IDEROPHORE PRODUCED BY P. fluorescens S HA 281 DIS S OLVED IN DMS O

FIG. (2) MASS SPECTRUM COLLISION ACTIVATION (MS-CA) OF [M+H]+ OF PYOVERDIN-S IDEROPHORE PRODUCED BY P. fluorescens S HA 281.

FIG. (3) THE PROPOS ED S TRUCTURE FOR PYOVERDINE PRODUCED BY P. fluorescens S HA 281.

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Characterization of the Siderophores-pyoverdine Produced by P. fluorescens SHA 281 1) Proton NMR Analysis of Purified Siderophorepyoverdine

determined based on the ladder protein markers.

The bands for samples ethyl acetate and purified by XAD-4 Amberlite resin were precipitated at one band with molecular weight ~12.5 kDa.

The basis for the sequence determination by NMR spectroscopy is the identification of all 1H of amino acids in the component. Proton nuclear magnetic resonance (P-NMR) analysis spectroscopy in Fig. (1) showed that, three amino acids were presented with different chemical shift δ (ppm) as appeared in Table (2). The following amino acids could be identified: Ser, Asp and Orn. TABLE (2) AMINO ACID SEQUENCE FROM

1H

NMR SPECTRA OF oC

PYOVERDINE -SIDEROPHORE DISSOLVED IN DMS O AT 30

N 1 2 3

Amino acid S er Asp Orn

Chemical shift δ (ppm) 3.399 2.502 2.083

2) Analysis of the Siderophores-pyoverdine Using Mass Spectrometry The amino acid sequence deduced from NMR data was confirmed by the fragment ions obtained after MS-CA spectra. The molecular mass of the main component was determined by MS-CA spectra at m/z 1049 as illustrated in Fig. (2 ). For structure elucidation, the precursor ions [M+H] + was excited

by CA in the ion trap of the mass spectrometer. The following amino acids could be identified: L-threoOHAsp, Gly, L-c(OH)Orn, 2 D-Ser, L-Thr and L-Val as appeared in Table (3).

TABLE (3) AMINO ACID SEQUENCE CHARACTERIZATION FRAGMENT IONS FOUND IN THE MS -CA SPECTRA OF [M+H] + FOR PYOVERDINE .

N 1 2 3 4 5 6 7

Amino acid S uc a-Chr-S er Val OHAsp Gly Thr S er c OHOrn+H+

m/z 444 543 674 731 832 919 1049

From the obtained results, the proposed structure for pyoverdine was appeared in Fig. (3).

3) Determination the Siderophore-pyoverdine

Molecular

Weight

of

Result in Fig. (4) showed the electrophoretic patterns of pyoverdine protein produced by P. fluorescens SAH 281. The analysis of SDS-PAGE for the extracted sample revealed the presence of bands with molecular weight similar to siderophore. Their values were 98

FIG. (4) AN 8% (v/v) S DS -POLYACRYLAMIDE GEL ELECTROPHORESIS OF PYOVERDINE PROTEIN PRODUCED BY Pseudompna s fluorescens S HA 281. LANE A: PYOVERDINE PROTEIN EXTRACTED BY ETHYL ACETATE; LANE B: PYOVERDINE PROTEIN PURIFIED BY XAD-4 AMBERLITE RES IN. M ILLUS TRATES THE PROTEIN LADDER WITH BAND S IZES S HOWN IN kDa. THE GEL WAS S TAINED WITH COOMAS S IE BRILLIANT BLUE R-250.

4) Identification of Siderophores-pyoverdine gene (PvdS) The PCR product was sequenced at GATC Biotech AG with ABI 3730 x l DNA Sequencing System using forward primer Fig.(5).

The resulting DNA sequence of the putative pvdS contained 492 bp compared to published sequences was made using the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov /blast), and investigated to homogeneity in the GenBank data base. The sequence of the eluted PCR product of Pseudomonas fluoresecens SHA 281 was 100 % homologs with the sequences of Pseudomonas fluoresecens F113 gb|CP000438.1| Length: |Len gth: 6845832: 2027261 to 2027752 of sigma factor PvdS as appeared in Fig.(6).

Through using search BLAST program to translate this sequence, it appeared that there was the homology of the translated sequencing of the deduced 492 bp to 164 amino acids of PvdS of P. fluoresecens SHA 281 as illustrated in Fig. (7,8).

Complexation with Siderophores-pyoverdine Produced by P. fluorescens SHA 281 It was appeared that, the highest percentage of uranium (VI) complexed with pyoverdine purified with XAD-4 Amberlite resin was (82.79 %), followed by extraction by ethyl acetate (79.81 % ) then crude pyoverdine in supernatant (77.60 %).

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1 61 121 181 241 301 361 421 481

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CCGCCGGGCGAAGGTATCTCCTCGAACCCCCGACACCTTGCGGCAATGCACCAGGGCATC 60 GCGGATCATGAAGTTCACCAGGGTCGGCGAAACGCCCAGCTCCTTGGCGATGTCCTTCTG 120 TGGCACGCCATGCAGGCGGTACATCTCGAAGGCATAGCGGGTGCGGCTGGGCAATTCGGT 180 CAGTGCGTCGGCGATGTGCTCCAGCGTGGAAAAGTTGATATGGGAGGTTTCTGGCGAAGC 240 ACCCTGGATCACCACGTTCAACCCTTCCTCTTCCGGCCCCGAGTATTTTTGTTCCAGCGC 300 CTGCTTGCGGTAGTGGTCGATCGCCAGGTTGCGCACGATCTGGAACAGGTAGCTGAGCTG 360 GGCCTTGAACGAGGAGGTGATCTGCGGCGCCGATTGCAGCCGGAAAAACGCGTCCTGCAC 420 CACATCTTCCGCGCGGGACCGACAGCCGGTAATGCGCGCCGCGATCTTGACCAGGATCAA 480 TCGGTTGTCCAC 492 FIG. (5) S EQUENCE OF THE PCR US ING FORWARD PRIMER 492 bp.

Query

1

Sbjct Query

2027261 61

Sbjct Query

2027321 121

Sbjct Query

2027381 181

Sbjct Query

2027441 241

Sbjct Query

2027501 301

Sbjct Query

2027561 361

Sbjct Query

2027621 421

Sbjct Query

2027681 481

Sbjct

2027741

CCGCCGGGCGAAGGTATCTCCTCGAACCCCCGACACCTTGCGGCAATGCACCAGGGCATC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCGCCGGGCGAAGGTATCTCCTCGAACCCCCGACACCTTGCGGCAATGCACCAGGGCATC GCGGATCATGAAGTTCACCAGGGTCGGCGAAACGCCCAGCTCCTTGGCGATGTCCTTCTG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGGATCATGAAGTTCACCAGGGTCGGCGAAACGCCCAGCTCCTTGGCGATGTCCTTCTG TGGCACGCCATGCAGGCGGTACATCTCGAAGGCATAGCGGGTGCGGCTGGGCAATTCGGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGGCACGCCATGCAGGCGGTACATCTCGAAGGCATAGCGGGTGCGGCTGGGCAATTCGGT CAGTGCGTCGGCGATGTGCTCCAGCGTGGAAAAGTTGATATGGGAGGTTTCTGGCGAAGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CAGTGCGTCGGCGATGTGCTCCAGCGTGGAAAAGTTGATATGGGAGGTTTCTGGCGAAGC ACCCTGGATCACCACGTTCAACCCTTCCTCTTCCGGCCCCGAGTATTTTTGTTCCAGCGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACCCTGGATCACCACGTTCAACCCTTCCTCTTCCGGCCCCGAGTATTTTTGTTCCAGCGC CTGCTTGCGGTAGTGGTCGATCGCCAGGTTGCGCACGATCTGGAACAGGTAGCTGAGCTG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTGCTTGCGGTAGTGGTCGATCGCCAGGTTGCGCACGATCTGGAACAGGTAGCTGAGCTG GGCCTTGAACGAGGAGGTGATCTGCGGCGCCGATTGCAGCCGGAAAAACGCGTCCTGCAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGCCTTGAACGAGGAGGTGATCTGCGGCGCCGATTGCAGCCGGAAAAACGCGTCCTGCAC CACATCTTCCGCGCGGGACCGACAGCCGGTAATGCGCGCCGCGATCTTGACCAGGATCAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CACATCTTCCGCGCGGGACCGACAGCCGGTAATGCGCGCCGCGATCTTGACCAGGATCAA TCGGTTGTCCAC 492 |||||||||||| TCGGTTGTCCAC 2027752

60 2027320 120 2027380 180 2027440 240 2027500 300 2027560 360 2027620 420 2027680 480 2027740

FIG. (6) PAIRWIS E COMPARIS ON 100 % HOMOLOGY BETWEEN THE S EQUENCE OF THE ELUTED PCR PRODUCT OF P. fluoresecens S HA 281 WITH THE S EQUENCES ID: gb|CP000438.1|Length: |Length: 6845832: 2027261 to 2027752 bp using BLAS T. Query

492

VDNRLILVKIAARITGCRSRAEDVVQDAFFRLQSAPQITSSFKAQLSYLFQIVRNLAIDH

313

Sbjct

1

MDNRLILVKIAARITGCRSRAEDVVQDAFFRLQSAPQITSSFKAQLSYLFQIVRNLAIDH

60

Query

312

YRKQALEQKYSGPEEEGLNVVIQGASPETSHINFSTLEHIADALTELPSRTRYAFEMYRL

133

Sbjct

61

YRKQALEQKYSGPEEEGLNVVIQGASPETSHINFSTLEHIADALTELPSRTRYAFEMYRL

120

Query

132

HGVPQKDIAKELGVSPTLVNFMIRDALVHCRKVSGVRGDTFARR

1

Sbjct

121

HGVPQKDIAKELGVSPTLVNFMIRDALVHCRKVSGVRGDTFARR

164

FIG. (7) ALIGNMENT 99 % HOMOLOGY OF LENGTH 164 AMINO ACID WITH PvdS of EXTRACYTOPLAS MIC FUNCTION S IGMA-70 FACTOR OF P. fluoresecens F113.

Fig. (8) Putative c onserved dominas have been detec ted in PvdS .

TABLE (4) CHEMICAL ANALYSIS OF MONAZITE AND (Th-U) CONCENTRATE

Samples

Elements (%) RE 2 O3 Fe 2 O3 MnO CaO MgO ThO2 UO2 Na 2O K2 O SiO2 Al2 O3 P 2O5 LOI

Monazite

55.8

0.8

UDL

0.5

0.3

4.5

0.4

0.2

0.1

1.6

UDL 27.7 8.1

(Th-U) concentrate

20.1

5.5

0.4

0.1

0.3

19.0

2.4

0.5

0.5

2.9

UDL

6.9 41.4

UDL: under detec tion limit. LOI: Loss of ignition,

99

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Factors Affecting Complexation of Uranium from the Bioleached Liquors of Monazite and (Th-U) Concentrate with Siderophore-pyoverdine The chemical structure of monazite and (Th-U) concentrate were shown in table (4).

% of complexation U with pyoverdine

100

Monazite (Th-U) concentrate

90 80 70

80 70 60 50 40 30 20 100

150

175 200 Shaking speeds (rpm)

250

50

FIG. (12) EFFECT OF S HAKING S PEEDS (rpm) ON COMPLEXATION OF URANIUM WITH PYROVERDINE.

40 30 20 25

50

90

120

150

FIG. (9) EFFECT OF DIFFERENT PYOVERDINE (mg/20ml) CONCENTRATION ON THE COMPLEXATION PERCENTAGE OF URANIUM. (Th-U) concentrate

% of complexation U with pyoverdine

100

Monazite

90 80 70

The optimum conditions for maximum complexation percentages of uranium from the bioleached liquors of monazite and (Th-U) concentrate with pyoverdine were found as follow: the pyoverdine concentration was 90 mg/20 ml dist. H2 O at pH 6.5 as in Fig. (9 & 10).

Also, 40 minutes shaking time and shaking speed 175 rpm were found to be the best, respectively as in Fig. (11& 12). The complexation percentages of monazite and (Th-U) concentrate with pyoverdine under the previous optimum conditions were found to be 90.48 % and 86.41 % form the bioleached liquors of monazite and (Th-U) concentrate, respectively.

60

Discussion

50 40 30 20 1.3 0.9 4 3.3 6.3 5.5 8.9 6.5 9.5 8.8 2.5

4.5

6.5

8.5

10.5

pH values

FIG. (10) EFFECT OF pH VALUES ON THE COMPLEXATION OF URANIUM WITH PYOVERDINE.

% of complexation U with pyoverdine

(Th-U) concentrate

90

60

Pyoverdine concentration (mg/20 ml) dist.water

Monazite

100

(Th-U) concentrate

90 80 70 60 \

50 40 30 20 10

20

30

40

50

Shaking times (min)

FIG. (11): EFFECT OF S HAKING TIMES (min) ON COMPLEXATION OF URANIUM WITH PYROVERDINE.

100

Monazite

100 % of complexation U with pyoverdine

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Initial excessive production of chelators may be crucial to further cell growth in the pesudomonads, as siderophore have already been released in the adaptation (lag) phase (Kalinowski et al., 2004). Th e pyoverdines were remarkably diverse, differing from one another by the number and types of amino acids in the peptide chain (Tripathi et al., 2005). Tang et al.

(2000) found that 16S rRNA gene sequence provided more rapid, unambiguous identification of the difficult bacterial isolates than conventional methods. PCR amplification of the 16S rRNA gene showed the same fragment for the sample confirming that it belongs to the same specie. Thus P. fluorescens SHA 281 cultivated in SA medium produced siderophore-pyoverdine (275 mg/l). Also, Nair et al. (2007) found that, P. azotoformans cultivated in succinic acid medium was found to produce mixed type of siderophore both catecholate and hydroxamate (280 mg/l). Mass spectroscopy and NMR analyses were used to determine the molecular weight and protons of the purified pyoverdine-siderophore extracted from P. fluorescens SHA 281. The extracted siderophorespyoverdine was observed at m/z 1049 with seven

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amino acids. This obtained result was similar to that obtained by (Ongena et al., 2001) who mentioned that the pyoverdine of P. fluorescens BTP2 with seven amino acids in its peptide chain belongs to the smallest representatives of its class. It is unique in its structure in so far that except for the Fe3+ chelating amino acids OHAsp and cOHOrn, and contains only small neutral ones, viz. Ser and Thr in addition to the unexpectedly lipophilic Val not encountered so far in pyoverdins.

The amino acid composition of pyoverdins serves several purposes: providing two of the ligand sites for Fe3+ and other ions in an appropriate position for the binding of this ion, and responsible for the recognition of the complex at the bacterial cell surface, frequently safeguarding that the own ferri-pyoverdin cannot be taken up by other strains. Protein banding pattern (SDS-PAGE) analysis of the extracted sample revealed the presence of band with molecular weight similar to siderophore determined based on the protein ladder markers. One band of protein was precipitated at ~30 kDa by ethyl acetate and XAD-4 Amberlite resin. This result was similar to that obtained by Dhanya and Potty (2007). According to their results P. fluorescens produced siderophores with molecular weights of 8.3, 9.5, 10.4 and 12.5 kDa. The result indicated that the sigma factor of PvdS regulates pyoverdine synthesis is conserved among the fluorescent pseudomonads. Th e amino acid sequence of the PvdS protein was similar (nearly 99% identity) to that of iron-responsive regulators from other fluorescent pseudomonads.

These proteins were also similar in function, all being involved in the transcriptional activation of genes for the biosynthesis of fluorescent siderophores (pyoverdine or pseudobactins) in Pseudomonas spp (Sexton et al., 1995). Also, pyoverdine pvdS from Pseudomonas fluorescens SHA 281 as 573-bp fragment containing the entire PvdS coding sequence was generated by PCR as mentioned by (Leoni et al., 2000).

The pyoverdine molecule denoted as LH4 , according to the general assumption that pyoverdine molecules can liberate four labile protons from the complexing sites most likely responsible for metal binding (Bouby et al., 1998). The formation of complexes of UO22+ with P. fluorescens SHA 281 pyoverdine was dependent on UO22+ concentration and pH as previously mentioned

by Moll et al. (2005).

The recommended optimum conditions for maximum complexation of U with siderophore-pyoverdine produced by P. fluorescens SHA 281 were in agreement with that previously recommended by Kalinowski et al.

(2006). Concerning the pH value the complexation

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remained high, up to pH 6.5. Then the complexation percentage gradually decreased as the pH value increased. This suggested the formation of third uranium (VI) pyoverdine-siderophore complex hydrolysis product as mentioned by Mullen et al.

(2007). Also, presence of Asp in the peptide chain was responsible for the acidic pH value as previously mentioned by Amann et al. (2000).

ACKNOWLEDGEMENTS

The authors gratefully acknowledgeed the financial support provided by the Zagazig University and Nuclear Materials Authority, Cairo, Egypt.

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