Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site

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Roth" "tienePdf" => "es" "tieneTextoCompleto" => "es" "tieneResumen" => array:2 [ 0 => "es" 1 => "en" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "47" "paginaFinal" => "55" ] ] "titulosAlternativos" => array:1 [ "en" => array:1 [ "titulo" => "Responses of the common bean (Phaseolus vulgaris L.) and Rhizobium tropici CIAT899 symbiosystem to induced allelopathy by Ipomoea purpurea L. Roth" ] ] "contieneResumen" => array:2 [ "es" => true "en" => true ] "contieneTextoCompleto" => array:1 [ "es" => true ] "contienePdf" => array:1 [ "es" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figura 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1775 "Ancho" => 1527 "Tamanyo" => 105396 ] ] "descripcion" => array:1 [ "es" => "Dinámica del crecimiento radical de plántulas de frijol germinadas en presencia de diferentes cantidades de lixiviados acuosos de raíz (A) y de parte aérea (B) de Ipomoea purpurea. Promedio±error estándar de la media. Letras distintas a un mismo tiempo indican diferencias significativas (Tukey, p=0,05)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Paulina Janneth Pérez-Peralta, Ronald Ferrera-Cerrato, Alejandro Alarcón, Libia I. Trejo-Téllez, Rocío Cruz-Ortega, Hilda V. Silva-Rojas" "autores" => array:6 [ 0 => array:2 [ "nombre" => "Paulina Janneth" "apellidos" => "Pérez-Peralta" ] 1 => array:2 [ "nombre" => "Ronald" "apellidos" => "Ferrera-Cerrato" ] 2 => array:2 [ "nombre" => "Alejandro" "apellidos" => "Alarcón" ] 3 => array:2 [ "nombre" => "Libia I." "apellidos" => "Trejo-Téllez" ] 4 => array:2 [ "nombre" => "Rocío" "apellidos" => "Cruz-Ortega" ] 5 => array:2 [ "nombre" => "Hilda V." 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Carbonari, Beatriz A. D’Astek, Gisela Zolezzi, Natalia Deza, Martin P. Palladino, Marcelo Masana, Isabel Chinen, Marta Rivas" "autores" => array:9 [ 0 => array:2 [ "nombre" => "Mariana" "apellidos" => "Cap" ] 1 => array:2 [ "nombre" => "Claudia C." "apellidos" => "Carbonari" ] 2 => array:2 [ "nombre" => "Beatriz A." "apellidos" => "D’Astek" ] 3 => array:2 [ "nombre" => "Gisela" "apellidos" => "Zolezzi" ] 4 => array:2 [ "nombre" => "Natalia" "apellidos" => "Deza" ] 5 => array:2 [ "nombre" => "Martin P." "apellidos" => "Palladino" ] 6 => array:2 [ "nombre" => "Marcelo" "apellidos" => "Masana" ] 7 => array:2 [ "nombre" => "Isabel" "apellidos" => "Chinen" ] 8 => array:2 [ "nombre" => "Marta" "apellidos" => "Rivas" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754118300518?idApp=UINPBA00004N" "url" => "/03257541/0000005100000001/v1_201903210629/S0325754118300518/v1_201903210629/en/main.assets" ] "en" => array:19 [ "idiomaDefecto" => true "cabecera" => "Original article" "titulo" => "Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "39" "paginaFinal" => "46" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Hamdy A. Hassan, Somya E. Dawah, Mostafa M. El-Sheekh" "autores" => array:3 [ 0 => array:4 [ "nombre" => "Hamdy A." "apellidos" => "Hassan" "email" => array:1 [ 0 => "hamdy.hassan@gebri.usc.edu.eg" ] "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0015" ] 2 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] 1 => array:3 [ "nombre" => "Somya E." "apellidos" => "Dawah" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "Mostafa M." "apellidos" => "El-Sheekh" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "c" "identificador" => "aff0010" ] ] ] ] "afiliaciones" => array:3 [ 0 => array:3 [ "entidad" => "Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research, Institute, University of Sadat City, Sadat City, Egypt" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Department of Biological Science, Faculty of Science and Humanity Studies at Al-Quwayiyah, Shaqra University, Al-Quwayiyah 11971, Saudi Arabia" "etiqueta" => "b" "identificador" => "aff0015" ] 2 => array:3 [ "entidad" => "Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt" "etiqueta" => "c" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Monitorización de la capacidad de degradación de las nuevas bacterias haloalcalifílicas resistentes a cloruro de tributiltina (TBTCl) en una ubicación contaminada por butiltina" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 1187 "Ancho" => 2333 "Tamanyo" => 224918 ] ] "descripcion" => array:1 [ "en" => "Phylogenetic tree showing the relatedness on the basis of TBTB permease. The dendrogram was calculated using Phylogeny.fr based on protein sequence alignments. TBTB permease proteins from Stenotrophomonas chelatiphaga HS2 are pointed by an arrow. The scale bar corresponds to an estimated evolutionary distance of 1 amino acid substitution per site." ] ] ] "textoCompleto" => "IntroductionTributyltin (TBT) is an organotin compound commonly used as fungicide, bactericide, pesticide, wood preservative, polyvinyl chloride (PVC) stabilizer and component of antifouling paints, which are used to coat structures exposed to aquatic environments, including ships, pleasure boats, buoys, pilings, sea walls, oil rigs, cables, and water intake pipes<a class="elsevierStyleCrossRef" href="#bib0235">12</a>. TBT repels or kills harassing organisms such as barnacles<a class="elsevierStyleCrossRef" href="#bib0280">21</a>.TBT can be highly toxic to many aquatic and terrestrial eukaryotic or prokaryotic organisms and causes immune system suppression and endocrine disruption in humans<a class="elsevierStyleCrossRef" href="#bib0230">11</a>. Generally, tributyltins (TBT) are more toxic than dibutyltin (DBT) and monobutyltin (MBT)<a class="elsevierStyleCrossRef" href="#bib0240">13</a>. Although the industries containing TBT have been banned by the International Maritime Organization<a class="elsevierStyleCrossRef" href="#bib0285">22</a>, some countries continued to use it as a biocide and wood preservative component. On the other hand, DBT and MBT have been found in drinking water<a class="elsevierStyleCrossRef" href="#bib0320">29</a>.Although TBT is toxic to both gram positive and gram negative bacteria collected from sediment, gram positive bacteria are generally more sensitive to TBT<a class="elsevierStyleCrossRef" href="#bib0200">5</a>. Bacterial TBT resistance can be achieved either by sequential removal of butyl groups from the tin atom, leading to the formation of DBT and MBT or pumping out TBT through a multidrug efflux pump<a class="elsevierStyleCrossRef" href="#bib0295">24</a> or bioaccumulation of TBT into the bacterial cell<a class="elsevierStyleCrossRef" href="#bib0250">15</a>. TBT can be degraded by gram negative or gram positive bacteria<a class="elsevierStyleCrossRefs" href="#bib0200">5,11</a>, whereas metabolic pathways of TBT by the reported bacteria have not been well studied and still requires elucidation of TBT degradation<a class="elsevierStyleCrossRef" href="#bib0315">28</a>.Haloalkaliphilic organisms are interesting from two points of view, fundamentally research and biotechnology<a class="elsevierStyleCrossRef" href="#bib0290">23</a>. Haloalkaliphilic degrading bacteria is an interesting class of extremophilic organisms that possess special adaptation strategies that make them interesting for the remediation of polluted sites<a class="elsevierStyleCrossRefs" href="#bib0270">19,32</a>. Moderate halophiles have the ability for exciting and promising applications making them the most potential candidates compared with other extremophiles<a class="elsevierStyleCrossRefs" href="#bib0265">18,27</a>.To our knowledge no haloalkaliphilic bacteria from the Mediterranean Sea have been previously isolated and reported to exhibit resistance or to possess TBT degradation ability.The objective of this study is to isolate and characterize haloalkaliphilic bacteria strains from the Mediterranean Sea, Abu Qir- coastline, Egypt, that have the ability to degrade TBT into DBT and MBT.Materials and methodsIsolation and identification of haloalkaliphilic bacteria degrading TBTWater and sediment samples contaminated with TBT were collected from the Mediterranean Sea, Abu Qir-coastline, Egypt. Samples were taken with pH 9 and salinity 32% (w/v) and placed in sterile bottles swabbed with 75% (v/v) ethanol, and stored at 4°C until they were used. Primary enrichment cultures were prepared in 100-ml bottles by adding 1ml of water sample to 19ml of mineral medium (MM)<a class="elsevierStyleCrossRef" href="#bib0220">9</a> containing 3% NaCl (w/v) and pH 9 (adjusted by adding Na2CO3) with TBT (125μM) as a sole source of carbon and energy. The enrichment cultures were incubated at 30°C; after one month 10% (v/v) of the culture was transferred to fresh MM containing 5% NaCl (w/v) and cultured for a further month and this step was repeated on 7% NaCl (w/v). Dilution of the cultures was spread onto MM agar plates supplemented with TBT and incubated for 7 days, the colonies observed were purified on MM agar plates with TBT. Three purified isolates (HS1, HS2 and HS5) were selected and identified by 16S rRNA. Three primers were used in the amplification of 16S rDNA, which included: Bact 27f (5′-AGAGTTTGATC(A/C)TGGCTCAG-3′), Bact 1492r (5′-TACGG(C/T)ACCTTGTTACGACTT-3′), and Bact 1098r (5′-AAGGGTTGCGCTCGTTGCG-3′)<a class="elsevierStyleCrossRef" href="#bib0185">2</a>. Theoretically, amplification with Bact 27f -1492r should yield 1505bp whereas amplification with Bact27f -1098r should yield 1108bp from the 16S rRNA. Amplifications with these two primer sets were used to obtain the nearly full-length sequence (1492bp) of the 16S rDNA of the isolate. PCR amplification was performed in a total volume of 5μl in a Touch Screen Thermal Cycler PCR Model: A100/A200 (Hangzhou LongGene Scientific Instruments Co., Ltd). The PCR product was purified by Gene JET™ Gel Extraction kit (Thermo Scientific) and sequenced in both directions. The determined 16S rRNA gene nucleotide sequences were entered for BLAST search into the NCBI website (<a id="intr0005" class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/blast/">http://www.ncbi.nlm.nih.gov/blast/</a>), and aligned using Clustal W implemented in MEGA software version 3, 1. The phylogenetic tree was constructed using Phylogeny.Fr<a class="elsevierStyleCrossRef" href="#bib0215">8</a>.TBT sensitivityHS1, HS2 and HS5 samples were spread on the surface of MM agar plates containing 125μM TBT. Plates were incubated at 30°C in the dark to avoid photodegradation of TBT. The isolates that have the ability to grow on 125μM TBT were successively transferred to MM plates containing increasingly higher TBT concentrations (0–3mM). Successive transferring to a new media occurred as long as growth was observed and the isolates were scored as resistant if they could grow along the gradient plates.Antibiotic susceptibilityAntibiotics to be tested were selected according to the Clinical Laboratory Standard Institute (CLSI)<a class="elsevierStyleCrossRef" href="#bib0195">4</a> guidelines, using the following antibiotic susceptibility discs supplied by Oxoid (Wade Rd, Basingstoke, Hampshire RG24 8PW, United Kingdom): Ampicillin 10μg/disc, ampicillin–sulbactam 10/10μg/disc, amoxicillin 10μg/disc, amoxicillin–clavulanic acid 30μg/disc, erythromycin 15μg/disc, azithromycin 15μg/disc, levofloxacin 5μg/disc, ciprofloxacin 5μg/disc, tetracycline10μg/disc, tigecycline 15μg/disc, clindamycin 2μg/disc, rifampicin 30μg/disc, septrin 25μg/disc, linezolid 30μg/disc, vancomycin 30μg/disc, oxacillin 1μg/disc, gentamicin 10μg/disc, and amikacin 30μg/disc.Effect of pH and salinity on TBT degrading abilityBacterial pure cultures from HS1, HS2 and HS5 were grown at 30°C with shaking (150rpm) in MM containing 2mM of TBT until late exponential growth phase. Cells were harvested by centrifugation, washed twice in sterile MM and resuspended to an OD600nm of 5. The effect of salt on TBT biodegradation was determined at various concentrations of 0–12% NaCl (w/v) by the methods described above. The effect of pH on TBT biodegradation was determined by using 50mM NaHCO3 (pH 7.0–9.0), 50mM Na2CO3 (pH 9–11). CFU/ml was monitored in sterile TBT-containing medium, and the residual TBT concentration of each compound from TBT separately was measured at regular intervals over an incubation period<a class="elsevierStyleCrossRefs" href="#bib0265">18,20</a>.Growth curve and TBT analysisTo quantify growth rate and substrate disappearance, Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5 were grown as described above and cultures were harvested during late exponential growth phase by centrifugation at 7000rpm for 10min. Cells were washed twice with 50mM phosphate buffer (pH 7) and resuspended in liquid MM to give an OD600nm of 0.1. Degradation of TBT was tested in sterilized glass tubes containing 2ml cell suspension (OD600nm=0.1) and 2mM of TBT as sole carbon source. The test tubes were incubated at 150rpm and 30°C. For estimation of the colony forming units (CFU), aliquots were serially diluted, 100μl aliquots were plated on solid LB medium and the CFUs were counted after 2 days of incubation at 30°C. Uninoculated tubes and tubes without substrate served as controls. For the TBT analysis, the sample vial was purged with nitrogen gas to achieve an inert atmosphere chamber to exclude oxygen and water. A 90μl solution of 0.8M n-hexylmagnesium bromide was added and derivatized for 30min. The reaction was stopped by adding 1ml of 2M HCl and the solution was set aside for 30min. The organic phase was separated by n-hexane and moisture was removed with anhydrous ammonium sulfate, 1μl was injected into gas chromatography (Agilent 7890A GC system) with a flame ionization detector (FID). GC analyses were performed under flow of nitrogen (2ml/min) on an HP-5 column (30m length, 0.25mm I.D., and 0.25μM film thickness). The oven temperature was increased from 80°C to 320°C by a rate of 3°C/min. TBT and DBT, purchased from Sigma Aldrich, (Germany) were injected into GC-FID used as standards according to their retention time and peak area.Amplification of TBT degrading geneGenomic DNA was extracted from strains HS1, HS2 and HS5 grown on MM+Na2CO3 (pH 10) and salinity 7% NaCl (w/v) in the presence of TBT as a sole carbon source. The isolated DNA was screened for the presence of tbt gene using specific primers designed based on the Av27-sugE gene, which was involved in TBT degradation by Aeromonas molluscorum Av27 (5′-ATGCCCTGGATATTGCTGCTC-3′ and 5′-GGGTGAAACCTTGGGTGTATTTG-3′)<a class="elsevierStyleCrossRef" href="#bib0210">7</a>. To validate that the correct PCR fragments had been amplified, the suspected band was purified, ligated into pGEM®-T Easy Vector system 1 (Promega) and cloned into Escherichia coli DH5α prepared as competent cells by transform aid bacterial transformation Kit and its protocol (Thermo Scientific). The transformed cells were plated on Luria-Bertani (LB) agar plates containing ampicillin (50μg/ml) and the plate was incubated at 37°C overnight. Positive colony plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Scientific) and the obtained gene was amplified by PCR using the M13f (5′-AGCGGATAACAATTTCACACAGGA-3′) and M13r (5′-CATTTTGCTGCCGGTCA-3′). The purified DNA was sequenced and the nucleotide sequences determined in this study were compared with existing sequences in GenBank by performing a BLASTn and BLASTp search.Extraction of mRNA, cDNA synthesis, and RT-PCRFor gene expression studies, S. chelatiphaga HS2 was grown on TBT (2mM) as the sole carbon source. To assess constitutive expression, the strain was grown in parallel on fructose (2mM). Cultures were harvested during exponential growth by centrifugation. Total RNA was isolated from 3ml of TBT or fructose-grown cells. Harvested cells were resuspended in 100μl water and immediately processed as previously described<a class="elsevierStyleCrossRef" href="#bib0345">34</a>. RNA was subsequently purified using the RNeasy kit (Qiagen), and 2μl of the eluted RNA (60μl) was separated in 1% (w/v) agarose gels and stained with ethidium bromide. cDNA was synthesized from 1μl of total RNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) from RNA templates. The kit uses RevertAid Reverse Transcriptase (RT), a recombinant M-MuLV RT which maintains activity at 42–50°C. RiboLock RNase Inhibitor supplied with the kit effectively protects RNA templates from degradation. The reverse transcription reaction mixtures were serially diluted (3.2-fold) with nuclease-free water (Qiagen), and 1μl of each dilution was subjected to amplification by PCR using the primer set TBTRNAF (5′-GATTCTTTGTGACGCGCCTT-3′) and TBTRNAR (5′-TCCCCATGTTATCCTCTGCC-3′) to amplify the TBTB permease gene fragment. Amplification products were separated on 1% (w/v) agarose gels and stained with ethidium bromide. Product bands were purified from agarose gels using a GeneJET PCR Purification Kit (Thermo Scientific) and sequenced to verify their identity.Nucleotide sequence accession numberThe 16S rRNA sequence for the reported strains in this study Sphingobium sp. HS1, S. chelatiphaga HS2 and R. borbori HS5, were deposited in the GenBank database under accession numbers KR780648, KR780649 and KR780650, respectively and the one corresponding to the TBTB permease under accession number KT590040.ResultsHaloalkaliphilic bacteria isolation and identificationIn this study three moderately haloalkaliphilic bacteria capable of growing on TBT as a sole source of carbon and energy were isolated from alkaline and saline water samples. The isolates could grow at pH 7.5–10, with optimum growth at pH 9 and 1.5–10% NaCl (w/v), with optimum growth at 7% NaCl (w/v). The three isolates could not grow at pH lower than 7 or salinity lower than 1.5% (w/v). 16S rRNA sequence results suggested that the isolates were phylogenetically most closely related to Sphingobium yanoikuyae with 97% similarity, S. chelatiphaga strain LPM-5 with 100% similarity and R. borbori strain DN316 with 99% similarity. The isolates were then classified as Sphingobium sp. HS1, S. chelatiphaga HS2 and R. borbori HS5, designated as HS1, HS2 and HS5, respectively and submitted to Genbank under accession numbers KR780648, KR780649 and KR780650.TBT sensitivity and degradation by the three identified haloalkaliphilic bacterial strainsTBT and its degradation products (DBT and MBT) were measured during the incubation of strains HS1, HS2 and HS5 for 40h in MM containing TBT concentrations (0–3mM), 7% NaCl (w/v) and pH 9. The negative control (the same media without inoculation) did not show TBT degradation. The results showed that strains HS1 and HS5 can grow at TBT concentrations (up to 1mM) while HS2 strain can grow at high TBT concentrations (up to 3mM) and its best growth was attained at 2mM TBT. Moreover, strains HS1, HS2 and HS5 showed a decrease in TBT percentage and generation of DBT and MBT as degradation products, indicating that these bacterial strains have the ability to reduce TBT concentration. No degradation occurred at NaCl concentrations above 10% (w/v), even after a higher incubation time, indicating that biodegradation inversely correlated with salinity at higher salt concentrations (data not shown).In strain HS1 about 41% of the original tin remained as TBT after 40h of incubation, 30% was converted into DBT and 20% into MBT and the remaining amount of TBT was converted into inorganic tin. In the case of strain HS5, the mean percentage of TBT that remained was almost 50%, after 40h of incubation, 25% was converted into DBT and 15% into MBT. According to the above results, strain HS1 and HS5 can be classified as moderate TBT degraders. Strain HS2 degraded more than 90% of TBT, converting 50% into DBT and 35% into MBT. The degradation proceeded according to the postulated scheme (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Finally, strain HS2 showed the highest TBT degradation capability, exceeding HS1 and HS5 abilities (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). Phylogenetic analysis of 16S rRNA gene for S. chelatiphaga HS2 (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>) clearly shows 100% similarity with S. chelatiphaga LPM-5 and 93% similarity with Stenotrophomonas maltophilia KB2.<elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia>The TBT degradation profile over 40h of incubation at 30°C, pH 9 and salinity 7% (w/v) for strain HS2 is shown in <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B. Between 10 and 30h a decrease in TBT concentration was observed, which was consistent with the rise in bacterial growth. Between 30 and 50h, the degradation rate declined along with the available nutrients, achieving more than 95% of degradation within 50h. The degradation of TBT contrasted with the generation of DBT and MBT as intermediate products. DBT concentration increased during incubation, reaching around 50% of the original TBT concentration (2mM), and MBT was detected with 35% during this period of incubation. The culture medium showed the brown, yellow and white colors that coincided with the postulated products that could be inorganic tin in the form of insoluble compounds (data not shown).Antibiotic susceptibility of S. chelatiphaga HS2Antibiotic susceptibility tests of strain HS2 showed that it was resistant to ampicillin, amoxicillin-clavulanic acid 30μg/disc as β-lactams antibiotics; however, it was susceptible to all other antibiotics tested.Detection and expression of a gene involved in tributyltin (TBT) resistance in S. chelatiphaga HS2In the present study, a gene was amplified using specific primers designed based on the TBT gene resistant sequence from TBT degrading bacterium A. molluscorum Av27<a class="elsevierStyleCrossRef" href="#bib0210">7</a>. The obtained fragment was cloned in E. coli DH5α using pGEM®-T Easy Vector. This clone contained pGEM®-T Easy vector with an inserted fragment of ∼725bp, whose deduced amino acid sequence has high homology (93% homology) with an aryl-sulfatase from S. maltophilia and (87% homology) with an arsenic efflux pump membrane protein from Xanthomonas oryzea and was identified as TBTB-permease (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>).<elsevierMultimedia ident="fig0020"></elsevierMultimedia>To verify whether the TBTB permease gene was expressed in response to TBT, RT-PCR experiments were performed with total RNA extracted from S. chelatiphaga HS2 growing on TBT and fructose. RT-PCR amplification products of the expected 400-bp size were observed with 3pg of RNA extracted from the culture grown on TBT (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>B), whereas no product was detected with RNA extracted from the culture grown on fructose (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>A), indicating that gene transcripts are induced at least 70-fold and also specifically induced in the presence of TBT. In addition, no amplification products were observed in controls devoid of reverse transcriptase or template cDNA (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>B lane C1′ and lane C2′). Sequencing of the approximately 400-bp product confirmed that it was identical to the corresponding TBTB permease gene fragment from genomic DNA of S. chelatiphaga HS2. These results showed direct evidence that the TBTB-permease gene product from S. chelatiphaga HS2 is definitely involved in TBT resistance.<elsevierMultimedia ident="fig0025"></elsevierMultimedia>DiscussionTributyltin (TBT) is considered a recalcitrant compound used as a biocide in the antifouling paints of boats and ship hulls, and then released slowly to the aquatic ecosystem in considerable amounts with toxic effect to a large number of aquatic organisms<a class="elsevierStyleCrossRef" href="#bib0190">3</a>. Despite the toxic effect of TBT on eukaryotic or prokaryotic organisms<a class="elsevierStyleCrossRef" href="#bib0180">1</a>, some microorganisms are resistant to TBT<a class="elsevierStyleCrossRef" href="#bib0350">35</a>. TBT degradation by the three strains decreased as salinity increased; furthermore, no degradation occurred at salinity greater than 10% NaCl (w/v) even with a long incubation time. This observation is consistent with those noted in extremely halotolerant bacteria<a class="elsevierStyleCrossRef" href="#bib0340">33</a>. Although much of the research on TBT degradation in terrestrial and marine environments has been studied extensively under oxic conditions<a class="elsevierStyleCrossRefs" href="#bib0210">7,24</a>, little is known about TBT degradation in hypersaline environments. Bioremediation of polluted hypersaline wastewaters and other environments with nonhalophilic microorganisms is difficult because salt inhibits their growth and the degradation of organic compounds<a class="elsevierStyleCrossRef" href="#bib0325">30</a>.The HS2 strain had higher ability to degrade TBT than HS1 and HS5 and showed 99% similarity with S. chelatiphaga LPM-5, the most closely related organism, which is an aerobic EDTA-degrading bacterium<a class="elsevierStyleCrossRef" href="#bib0300">25</a> and 93% with S. maltophilia KB2, which is a triphenyltin-degrading bacterium<a class="elsevierStyleCrossRef" href="#bib0255">16</a>. The HS2 strain has the ability to use TBT as carbon source in a mineral salt medium with 7% salinity and pH 9. Furthermore, TBT degraders A. molluscorum Av27 and Aeromonas veronii Av27, had also the capacity to use TBT as carbon source in a mineral salt medium<a class="elsevierStyleCrossRef" href="#bib0200">5</a> and showed optimum growth with salinity 4% (w/v) and pH 9<a class="elsevierStyleCrossRef" href="#bib0205">6</a>.Antibiotic test results confirm that the HS2 strain does not exhibit potential risk of toxicity and could be used for TBT bioremediation. The resistance of common antibiotics could be toxic and affect human health<a class="elsevierStyleCrossRef" href="#bib0245">14</a>. Moreover, the rapid appearance of antibiotic resistance among environmental bacteria as a result of horizontal gene transfer through plasmids, transposons and integrons is a known process<a class="elsevierStyleCrossRef" href="#bib0260">17</a>.To our knowledge, this is the first reported halophilic and alkaliphilic bacteria from the Mediterranean Sea that has the ability to degrade TBT under both halophilic and alkaliphilic conditions. The results suggested that S. chelatiphaga HS2 degraded TBT into DBT and MBT as less toxic compounds and this haloalkaliphilic strain could be applied in the remediation of TBT contaminants or natural attenuation of extreme alkaline and hypersaline TBT contaminated sites.TBT degraders received great attention, especially genes, pathways, and mechanisms of their degradation; however, until now the cellular and molecular mechanisms involved in TBT resistance are not clear and they are supposed to be different either among bacterial genera or even the same species or strain<a class="elsevierStyleCrossRefs" href="#bib0210">7,24</a>. Furthermore, it is not known whether haloalkaliphilic bacteria degrade TBT using novel genes and pathways compared to those used by non haloalkaliphiles. HS2 harbor the TBTB-permease gene which is a single, non-redundant, protein sequence, where anion permease like ArsB has been shown to export arsenate and antimonite in eubacteria and archaea. A typical ArsB permease contains 8–13 transmembrane helices and can function as a chemiosmotic transporter or as a channel-forming subunit of an ATP-driven anion pump. ArsB proteins belong to the ArsB/NhaD superfamily of permeases that translocate sodium, arsenate, sulfate, and organic anions across biological membranes<a class="elsevierStyleCrossRef" href="#bib0330">31</a>. Most metal resistance systems included tin accomplished by ATPase or a single polypeptide like (ArsB) efflux systems<a class="elsevierStyleCrossRef" href="#bib0225">10</a>. These systems could explain the ability of TBT resistant bacteria, either by formation of DBT and MBT as a result of removing butyl groups from the tin atom<a class="elsevierStyleCrossRef" href="#bib0200">5</a>, or pumping out TBT through a multidrug efflux pump<a class="elsevierStyleCrossRef" href="#bib0295">24</a> or TBT bioaccumulation in the bacterial cells<a class="elsevierStyleCrossRef" href="#bib0230">11</a>.This work presented the important role of TBT-degrading bacteria isolated from the Mediterranean Sea at a TBT-contaminated site, where three halophilic and alkaliphilic bacterial strains Sphingobium sp. HS1, S. chelatiphaga HS2 and R. borbori HS5 were isolated, having the ability to use TBT as a carbon source in minimal media with 7% (w/v) salinity and pH 9.S. chelatiphaga HS2 was the most efficient TBT degrader and had the ability to transform most of the TBT into DBT and MBT. In TBT-resistant bacterium S. chelatiphaga HS2, a new gene was identified as TBTB-permease, which had 93% similarity with aryl sulfatase in S. maltophilia and 87% similarity with arsenic efflux pump membrane protein in X. oryzea. The TBTB-permease gene was only expressed in the presence of TBT. The three novel haloalkaliphilic strains and their TBT degradation ability have never been reported previously. Moreover, HS2 strain is an effective candidate bacterium for TBT bioremediation in halophilic and alkaliphilic TBT contaminated sites.Conflict of interestThe authors declare that they have no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres1167518" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1092322" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres1167519" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec1092323" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:8 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Isolation and identification of haloalkaliphilic bacteria degrading TBT" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "TBT sensitivity" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Antibiotic susceptibility" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Effect of pH and salinity on TBT degrading ability" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Growth curve and TBT analysis" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Amplification of TBT degrading gene" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Extraction of mRNA, cDNA synthesis, and RT-PCR" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Nucleotide sequence accession number" ] ] ] 6 => array:3 [ "identificador" => "sec0055" "titulo" => "Results" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0060" "titulo" => "Haloalkaliphilic bacteria isolation and identification" ] 1 => array:2 [ "identificador" => "sec0065" "titulo" => "TBT sensitivity and degradation by the three identified haloalkaliphilic bacterial strains" ] 2 => array:2 [ "identificador" => "sec0070" "titulo" => "Antibiotic susceptibility of S. chelatiphaga HS2" ] 3 => array:2 [ "identificador" => "sec0075" "titulo" => "Detection and expression of a gene involved in tributyltin (TBT) resistance in S. chelatiphaga HS2" ] ] ] 7 => array:2 [ "identificador" => "sec0080" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0085" "titulo" => "Conflict of interest" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2017-08-03" "fechaAceptado" => "2017-12-11" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1092322" "palabras" => array:4 [ 0 => "Tributyltin" 1 => "Biodegradation" 2 => "Haloalkaliphilic" 3 => "ArsB permease" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1092323" "palabras" => array:4 [ 0 => "Tributiltina" 1 => "Biodegradación" 2 => "Haloalcalifílico" 3 => "ArsB permeasa" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "Tributyltin (TBT) is recognized as a major environmental problem at a global scale. Haloalkaliphilic tributyltin (TBT)-degrading bacteria may be a key factor in the remediation of TBT polluted sites. In this work, three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea. After analysis of the 16S rRNA gene sequences the isolates were identified as Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5. The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7% (w/v) salt concentration. S. chelatiphaga HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin (DBT and MBT). A gene was amplified from strain HS2 and identified as TBTB-permease-like, that encodes an ArsB-permease. A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance. The three novel haloalkaliphilic TBT degraders have never been reported previously." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "Se considera a la tributiltina (TBT) como un problema medioambiental serio a escala global. Las bacterias haloalcalifílicas degradadoras de TBT pueden constituir un factor clave para remediar áreas contaminadas con dicho xenobiótico. En este estudio se aislaron 3 cepas de bacterias haloalcalifílicas procedentes de un sitio contaminado con TBT en el mar Mediterráneo. Tras analizar las secuencias del gen de 16S del ARNr, se identificaron los aislados como Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 y Rhizobium borbori HS5. Las condiciones de crecimiento óptimas para la biodegradación de TBT por parte de las 3 cepas fueron pH 9 y 7% (p/v) de concentración de sal. S. chelatiphaga HS2 fue el degradador de TBT más efectivo, con capacidad de transformar la mayor parte de ese compuesto en dibutiltina y monobutiltina (DBT y MBT). Se amplificó un gen de la cepa HS2, que fue identificado como tipo TBTB-permeasa, que codifica para una ArsB permeasa. Un análisis de la cepa HS2 por reacción en cadena de la polimerasa con transcriptasa inversa (RT PCR) confirmó que el gen TBTB-permeasa contribuye a la resistencia al TBT. Estos 3 nuevos degradadores haloalcalifílicos de TBT no habían sido reportados previamente." ] ] "multimedia" => array:5 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 582 "Ancho" => 2328 "Tamanyo" => 57497 ] ] "descripcion" => array:1 [ "en" => "Proposed TBT debutylation pathway based on this study and other studies<a class="elsevierStyleCrossRefs" href="#bib0305">26,35</a>." ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 724 "Ancho" => 2386 "Tamanyo" => 169433 ] ] "descripcion" => array:1 [ "en" => "Growth of Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5 on 2mM TBT as a carbon source. Growth was monitored by following colony-forming units (CFU), TBT depletion and formation of DBT and MBT were assessed by GC." ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 765 "Ancho" => 2079 "Tamanyo" => 129852 ] ] "descripcion" => array:1 [ "en" => "Phylogenetic tree based on 16S rRNA gene sequences showing the relationship with Stenotrophomonas chelatiphaga. HS2 strain (accession number KR780649) pointed by an arrow to other reported Stenotrophomonas strains. The strain followed by the accession numbers and degraded substrate; petroleum hydrocarbon (petroleum H), benzene (BTEX), toluene, ethylbenzene and xylenes, triphenyltin (TPT), ethylenediaminetetra acetic acid (EDTA), and tributyltin (TBT) degraders." ] ] 3 => array:7 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 1187 "Ancho" => 2333 "Tamanyo" => 224918 ] ] "descripcion" => array:1 [ "en" => "Phylogenetic tree showing the relatedness on the basis of TBTB permease. The dendrogram was calculated using Phylogeny.fr based on protein sequence alignments. TBTB permease proteins from Stenotrophomonas chelatiphaga HS2 are pointed by an arrow. The scale bar corresponds to an estimated evolutionary distance of 1 amino acid substitution per site." ] ] 4 => array:7 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1337 "Ancho" => 2083 "Tamanyo" => 134192 ] ] "descripcion" => array:1 [ "en" => "RT-PCR amplification of TBTB permease mRNA from S. chelatiphaga HS2 grown on fructose (A) or TBT (B). M lanes contain the molecular size marker Hyperladder 1 (Bioline). cDNA generated from 1μg template RNA was serially diluted (3.2-fold) with nuclease-free water, and 1μl of each dilution was subjected to amplification by PCR (lanes 1 to 6) for cDNA from fructose and (lanes 1′ to 5′) for cDNA from TBT. Negative controls included undiluted RT-PCR mixtures devoid of reverse transcriptase (lane C1′) or template cDNA (lane C2′). 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Monitorización de la capacidad de degradación de las nuevas bacterias haloalcalifílicas resistentes a cloruro de tributiltina (TBTCl) en una ubicación contaminada por butiltina

Hamdy A. Hassana,b,

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hamdy.hassan@gebri.usc.edu.eg

Corresponding author.

, Somya E. Dawahc, Mostafa M. El-Sheekhc

a Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research, Institute, University of Sadat City, Sadat City, Egypt

b Department of Biological Science, Faculty of Science and Humanity Studies at Al-Quwayiyah, Shaqra University, Al-Quwayiyah 11971, Saudi Arabia

c Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt

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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Tributyltin &#40;TBT&#41; is an organotin compound commonly used as fungicide&#44; bactericide&#44; pesticide&#44; wood preservative&#44; polyvinyl chloride &#40;PVC&#41; stabilizer and component of antifouling paints&#44; which are used to coat structures exposed to aquatic environments&#44; including ships&#44; pleasure boats&#44; buoys&#44; pilings&#44; sea walls&#44; oil rigs&#44; cables&#44; and water intake pipes<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">12</span></a>&#46; TBT repels or kills harassing organisms such as barnacles<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">21</span></a>&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">TBT can be highly toxic to many aquatic and terrestrial eukaryotic or prokaryotic organisms and causes immune system suppression and endocrine disruption in humans<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">11</span></a>&#46; Generally&#44; tributyltins &#40;TBT&#41; are more toxic than dibutyltin &#40;DBT&#41; and monobutyltin &#40;MBT&#41;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">13</span></a>&#46; Although the industries containing TBT have been banned by the International Maritime Organization<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">22</span></a>&#44; some countries continued to use it as a biocide and wood preservative component&#46; On the other hand&#44; DBT and MBT have been found in drinking water<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">29</span></a>&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Although TBT is toxic to both gram positive and gram negative bacteria collected from sediment&#44; gram positive bacteria are generally more sensitive to TBT<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">5</span></a>&#46; Bacterial TBT resistance can be achieved either by sequential removal of butyl groups from the tin atom&#44; leading to the formation of DBT and MBT or pumping out TBT through a multidrug efflux pump<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">24</span></a> or bioaccumulation of TBT into the bacterial cell<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">15</span></a>&#46; TBT can be degraded by gram negative or gram positive bacteria<a class="elsevierStyleCrossRefs" href="#bib0200"><span class="elsevierStyleSup">5&#44;11</span></a>&#44; whereas metabolic pathways of TBT by the reported bacteria have not been well studied and still requires elucidation of TBT degradation<a class="elsevierStyleCrossRef" href="#bib0315"><span class="elsevierStyleSup">28</span></a>&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Haloalkaliphilic organisms are interesting from two points of view&#44; fundamentally research and biotechnology<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">23</span></a>&#46; Haloalkaliphilic degrading bacteria is an interesting class of extremophilic organisms that possess special adaptation strategies that make them interesting for the remediation of polluted sites<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">19&#44;32</span></a>&#46; Moderate halophiles have the ability for exciting and promising applications making them the most potential candidates compared with other extremophiles<a class="elsevierStyleCrossRefs" href="#bib0265"><span class="elsevierStyleSup">18&#44;27</span></a>&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">To our knowledge no haloalkaliphilic bacteria from the Mediterranean Sea have been previously isolated and reported to exhibit resistance or to possess TBT degradation ability&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">The objective of this study is to isolate and characterize haloalkaliphilic bacteria strains from the Mediterranean Sea&#44; Abu Qir- coastline&#44; Egypt&#44; that have the ability to degrade TBT into DBT and MBT&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Isolation and identification of haloalkaliphilic bacteria degrading TBT</span><p id="par0035" class="elsevierStylePara elsevierViewall">Water and sediment samples contaminated with TBT were collected from the Mediterranean Sea&#44; Abu Qir-coastline&#44; Egypt&#46; Samples were taken with pH 9 and salinity 32&#37; &#40;w&#47;v&#41; and placed in sterile bottles swabbed with 75&#37; &#40;v&#47;v&#41; ethanol&#44; and stored at 4<span class="elsevierStyleHsp" style=""></span>&#176;C until they were used&#46; Primary enrichment cultures were prepared in 100-ml bottles by adding 1<span class="elsevierStyleHsp" style=""></span>ml of water sample to 19<span class="elsevierStyleHsp" style=""></span>ml of mineral medium &#40;MM&#41;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">9</span></a> containing 3&#37; NaCl &#40;w&#47;v&#41; and pH 9 &#40;adjusted by adding Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span>&#41; with TBT &#40;125<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; as a sole source of carbon and energy&#46; The enrichment cultures were incubated at 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#59; after one month 10&#37; &#40;v&#47;v&#41; of the culture was transferred to fresh MM containing 5&#37; NaCl &#40;w&#47;v&#41; and cultured for a further month and this step was repeated on 7&#37; NaCl &#40;w&#47;v&#41;&#46; Dilution of the cultures was spread onto MM agar plates supplemented with TBT and incubated for 7 days&#44; the colonies observed were purified on MM agar plates with TBT&#46; Three purified isolates &#40;HS1&#44; HS2 and HS5&#41; were selected and identified by 16S rRNA&#46; Three primers were used in the amplification of 16S rDNA&#44; which included&#58; Bact 27f &#40;5&#8242;-AGAGTTTGATC&#40;A&#47;C&#41;TGGCTCAG-3&#8242;&#41;&#44; Bact 1492r &#40;5&#8242;-TACGG&#40;C&#47;T&#41;ACCTTGTTACGACTT-3&#8242;&#41;&#44; and Bact 1098r &#40;5&#8242;-AAGGGTTGCGCTCGTTGCG-3&#8242;&#41;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">2</span></a>&#46; Theoretically&#44; amplification with Bact 27f -1492r should yield 1505<span class="elsevierStyleHsp" style=""></span>bp whereas amplification with Bact27f -1098r should yield 1108<span class="elsevierStyleHsp" style=""></span>bp from the 16S rRNA&#46; Amplifications with these two primer sets were used to obtain the nearly full-length sequence &#40;1492<span class="elsevierStyleHsp" style=""></span>bp&#41; of the 16S rDNA of the isolate&#46; PCR amplification was performed in a total volume of 5<span class="elsevierStyleHsp" style=""></span>&#956;l in a Touch Screen Thermal Cycler PCR Model&#58; A100&#47;A200 &#40;Hangzhou LongGene Scientific Instruments Co&#46;&#44; Ltd&#41;&#46; The PCR product was purified by Gene JET&#8482; Gel Extraction kit &#40;Thermo Scientific&#41; and sequenced in both directions&#46; The determined 16S rRNA gene nucleotide sequences were entered for BLAST search into the NCBI website &#40;<a id="intr0005" class="elsevierStyleInterRef" href="http://www.ncbi.nlm.nih.gov/blast/">http&#58;&#47;&#47;www&#46;ncbi&#46;nlm&#46;nih&#46;gov&#47;blast&#47;</a>&#41;&#44; and aligned using Clustal W implemented in MEGA software version 3&#44; 1&#46; The phylogenetic tree was constructed using Phylogeny&#46;Fr<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">8</span></a>&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">TBT sensitivity</span><p id="par0040" class="elsevierStylePara elsevierViewall">HS1&#44; HS2 and HS5 samples were spread on the surface of MM agar plates containing 125<span class="elsevierStyleHsp" style=""></span>&#956;M TBT&#46; Plates were incubated at 30<span class="elsevierStyleHsp" style=""></span>&#176;C in the dark to avoid photodegradation of TBT&#46; 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septrin 25<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#44; linezolid 30<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#44; vancomycin 30<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#44; oxacillin 1<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#44; gentamicin 10<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#44; and amikacin 30<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Effect of pH and salinity on TBT degrading ability</span><p id="par0050" class="elsevierStylePara elsevierViewall">Bacterial pure cultures from HS1&#44; HS2 and HS5 were grown at 30<span class="elsevierStyleHsp" style=""></span>&#176;C with shaking &#40;150<span class="elsevierStyleHsp" style=""></span>rpm&#41; in MM containing 2<span class="elsevierStyleHsp" style=""></span>mM of TBT until late exponential growth phase&#46; Cells were harvested by centrifugation&#44; washed twice in sterile MM and resuspended to an OD<span class="elsevierStyleInf">600<span class="elsevierStyleHsp" style=""></span>nm</span> of 5&#46; The effect of salt on TBT biodegradation was determined at various concentrations of 0&#8211;12&#37; NaCl &#40;w&#47;v&#41; by the methods described above&#46; The effect of pH on TBT biodegradation was determined by using 50<span class="elsevierStyleHsp" style=""></span>mM NaHCO<span class="elsevierStyleInf">3</span> &#40;pH 7&#46;0&#8211;9&#46;0&#41;&#44; 50<span class="elsevierStyleHsp" style=""></span>mM Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span> &#40;pH 9&#8211;11&#41;&#46; CFU&#47;ml was monitored in sterile TBT-containing medium&#44; and the residual TBT concentration of each compound from TBT separately was measured at regular intervals over an incubation period<a class="elsevierStyleCrossRefs" href="#bib0265"><span class="elsevierStyleSup">18&#44;20</span></a>&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Growth curve and TBT analysis</span><p id="par0055" class="elsevierStylePara elsevierViewall">To quantify growth rate and substrate disappearance&#44; <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">Rhizobium borbori</span> HS5 were grown as described above and cultures were harvested during late exponential growth phase by centrifugation at 7000<span class="elsevierStyleHsp" style=""></span>rpm for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Cells were washed twice with 50<span class="elsevierStyleHsp" style=""></span>mM phosphate buffer &#40;pH 7&#41; and resuspended in liquid MM to give an OD<span class="elsevierStyleInf">600<span class="elsevierStyleHsp" style=""></span>nm</span> of 0&#46;1&#46; Degradation of TBT was tested in sterilized glass tubes containing 2<span class="elsevierStyleHsp" style=""></span>ml cell suspension &#40;OD<span class="elsevierStyleInf">600<span class="elsevierStyleHsp" style=""></span>nm</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;1&#41; and 2<span class="elsevierStyleHsp" style=""></span>mM of TBT as sole carbon source&#46; The test tubes were incubated at 150<span class="elsevierStyleHsp" style=""></span>rpm and 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; For estimation of the colony forming units &#40;CFU&#41;&#44; aliquots were serially diluted&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;l aliquots were plated on solid LB medium and the CFUs were counted after 2 days of incubation at 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Uninoculated tubes and tubes without substrate served as controls&#46; For the TBT analysis&#44; the sample vial was purged with nitrogen gas to achieve an inert atmosphere chamber to exclude oxygen and water&#46; A 90<span class="elsevierStyleHsp" style=""></span>&#956;l solution of 0&#46;8<span class="elsevierStyleHsp" style=""></span>M <span class="elsevierStyleItalic">n</span>-hexylmagnesium bromide was added and derivatized for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The reaction was stopped by adding 1<span class="elsevierStyleHsp" style=""></span>ml of 2<span class="elsevierStyleHsp" style=""></span>M HCl and the solution was set aside for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The organic phase was separated by <span class="elsevierStyleItalic">n</span>-hexane and moisture was removed with anhydrous ammonium sulfate&#44; 1<span class="elsevierStyleHsp" style=""></span>&#956;l was injected into gas chromatography &#40;Agilent 7890A GC system&#41; with a flame ionization detector &#40;FID&#41;&#46; GC analyses were performed under flow of nitrogen &#40;2<span class="elsevierStyleHsp" style=""></span>ml&#47;min&#41; on an HP-5 column &#40;30<span class="elsevierStyleHsp" style=""></span>m length&#44; 0&#46;25<span class="elsevierStyleHsp" style=""></span>mm I&#46;D&#46;&#44; and 0&#46;25<span class="elsevierStyleHsp" style=""></span>&#956;M film thickness&#41;&#46; The oven temperature was increased from 80<span class="elsevierStyleHsp" style=""></span>&#176;C to 320<span class="elsevierStyleHsp" style=""></span>&#176;C by a rate of 3<span class="elsevierStyleHsp" style=""></span>&#176;C&#47;min&#46; TBT and DBT&#44; purchased from Sigma Aldrich&#44; &#40;Germany&#41; were injected into GC-FID used as standards according to their retention time and peak area&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Amplification of TBT degrading gene</span><p id="par0060" class="elsevierStylePara elsevierViewall">Genomic DNA was extracted from strains HS1&#44; HS2 and HS5 grown on MM<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span> &#40;pH 10&#41; and salinity 7&#37; NaCl &#40;w&#47;v&#41; in the presence of TBT as a sole carbon source&#46; The isolated DNA was screened for the presence of <span class="elsevierStyleItalic">tbt</span> gene using specific primers designed based on the Av27-sugE gene&#44; which was involved in TBT degradation by <span class="elsevierStyleItalic">Aeromonas molluscorum</span> Av27 &#40;5&#8242;-ATGCCCTGGATATTGCTGCTC-3&#8242; and 5&#8242;-GGGTGAAACCTTGGGTGTATTTG-3&#8242;&#41;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">7</span></a>&#46; To validate that the correct PCR fragments had been amplified&#44; the suspected band was purified&#44; ligated into pGEM<span class="elsevierStyleSup">&#174;</span>-T Easy Vector system 1 &#40;Promega&#41; and cloned into <span class="elsevierStyleItalic">Escherichia coli</span> DH5&#945; prepared as competent cells by transform aid bacterial transformation Kit and its protocol &#40;Thermo Scientific&#41;&#46; The transformed cells were plated on Luria-Bertani &#40;LB&#41; agar plates containing ampicillin &#40;50<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml&#41; and the plate was incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C overnight&#46; Positive colony plasmids were isolated using the GeneJET Plasmid Miniprep Kit &#40;Thermo Scientific&#41; and the obtained gene was amplified by PCR using the M13f &#40;5&#8242;-AGCGGATAACAATTTCACACAGGA-3&#8242;&#41; and M13r &#40;5&#8242;-CATTTTGCTGCCGGTCA-3&#8242;&#41;&#46; The purified DNA was sequenced and the nucleotide sequences determined in this study were compared with existing sequences in GenBank by performing a BLASTn and BLASTp search&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Extraction of mRNA&#44; cDNA synthesis&#44; and RT-PCR</span><p id="par0065" class="elsevierStylePara elsevierViewall">For gene expression studies&#44; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 was grown on TBT &#40;2<span class="elsevierStyleHsp" style=""></span>mM&#41; as the sole carbon source&#46; To assess constitutive expression&#44; the strain was grown in parallel on fructose &#40;2<span class="elsevierStyleHsp" style=""></span>mM&#41;&#46; Cultures were harvested during exponential growth by centrifugation&#46; Total RNA was isolated from 3<span class="elsevierStyleHsp" style=""></span>ml of TBT or fructose-grown cells&#46; Harvested cells were resuspended in 100<span class="elsevierStyleHsp" style=""></span>&#956;l water and immediately processed as previously described<a class="elsevierStyleCrossRef" href="#bib0345"><span class="elsevierStyleSup">34</span></a>&#46; RNA was subsequently purified using the RNeasy kit &#40;Qiagen&#41;&#44; and 2<span class="elsevierStyleHsp" style=""></span>&#956;l of the eluted RNA &#40;60<span class="elsevierStyleHsp" style=""></span>&#956;l&#41; was separated in 1&#37; &#40;w&#47;v&#41; agarose gels and stained with ethidium bromide&#46; cDNA was synthesized from 1<span class="elsevierStyleHsp" style=""></span>&#956;l of total RNA using a RevertAid First Strand cDNA Synthesis Kit &#40;Thermo Scientific&#41; from RNA templates&#46; The kit uses RevertAid Reverse Transcriptase &#40;RT&#41;&#44; a recombinant M-MuLV RT which maintains activity at 42&#8211;50<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; RiboLock RNase Inhibitor supplied with the kit effectively protects RNA templates from degradation&#46; The reverse transcription reaction mixtures were serially diluted &#40;3&#46;2-fold&#41; with nuclease-free water &#40;Qiagen&#41;&#44; and 1<span class="elsevierStyleHsp" style=""></span>&#956;l of each dilution was subjected to amplification by PCR using the primer set TBTRNAF &#40;5&#8242;-GATTCTTTGTGACGCGCCTT-3&#8242;&#41; and TBTRNAR &#40;5&#8242;-TCCCCATGTTATCCTCTGCC-3&#8242;&#41; to amplify the TBTB permease gene fragment&#46; Amplification products were separated on 1&#37; &#40;w&#47;v&#41; agarose gels and stained with ethidium bromide&#46; Product bands were purified from agarose gels using a GeneJET PCR Purification Kit &#40;Thermo Scientific&#41; and sequenced to verify their identity&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Nucleotide sequence accession number</span><p id="par0070" class="elsevierStylePara elsevierViewall">The 16S rRNA sequence for the reported strains in this study <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">R&#46; borbori</span> HS5&#44; were deposited in the GenBank database under accession numbers KR780648&#44; KR780649 and KR780650&#44; respectively and the one corresponding to the TBTB permease under accession number KT590040&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Haloalkaliphilic bacteria isolation and identification</span><p id="par0075" class="elsevierStylePara elsevierViewall">In this study three moderately haloalkaliphilic bacteria capable of growing on TBT as a sole source of carbon and energy were isolated from alkaline and saline water samples&#46; The isolates could grow at pH 7&#46;5&#8211;10&#44; with optimum growth at pH 9 and 1&#46;5&#8211;10&#37; NaCl &#40;w&#47;v&#41;&#44; with optimum growth at 7&#37; NaCl &#40;w&#47;v&#41;&#46; The three isolates could not grow at pH lower than 7 or salinity lower than 1&#46;5&#37; &#40;w&#47;v&#41;&#46; 16S rRNA sequence results suggested that the isolates were phylogenetically most closely related to <span class="elsevierStyleItalic">Sphingobium yanoikuyae</span> with 97&#37; similarity&#44; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> strain LPM-5 with 100&#37; similarity and <span class="elsevierStyleItalic">R&#46; borbori</span> strain DN316 with 99&#37; similarity&#46; The isolates were then classified as <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">R&#46; borbori</span> HS5&#44; designated as HS1&#44; HS2 and HS5&#44; respectively and submitted to Genbank under accession numbers KR780648&#44; KR780649 and KR780650&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">TBT sensitivity and degradation by the three identified haloalkaliphilic bacterial strains</span><p id="par0080" class="elsevierStylePara elsevierViewall">TBT and its degradation products &#40;DBT and MBT&#41; were measured during the incubation of strains HS1&#44; HS2 and HS5 for 40<span class="elsevierStyleHsp" style=""></span>h in MM containing TBT concentrations &#40;0&#8211;3<span class="elsevierStyleHsp" style=""></span>mM&#41;&#44; 7&#37; NaCl &#40;w&#47;v&#41; and pH 9&#46; The negative control &#40;the same media without inoculation&#41; did not show TBT degradation&#46; The results showed that strains HS1 and HS5 can grow at TBT concentrations &#40;up to 1<span class="elsevierStyleHsp" style=""></span>mM&#41; while HS2 strain can grow at high TBT concentrations &#40;up to 3<span class="elsevierStyleHsp" style=""></span>mM&#41; and its best growth was attained at 2<span class="elsevierStyleHsp" style=""></span>mM TBT&#46; Moreover&#44; strains HS1&#44; HS2 and HS5 showed a decrease in TBT percentage and generation of DBT and MBT as degradation products&#44; indicating that these bacterial strains have the ability to reduce TBT concentration&#46; No degradation occurred at NaCl concentrations above 10&#37; &#40;w&#47;v&#41;&#44; even after a higher incubation time&#44; indicating that biodegradation inversely correlated with salinity at higher salt concentrations &#40;data not shown&#41;&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">In strain HS1 about 41&#37; of the original tin remained as TBT after 40<span class="elsevierStyleHsp" style=""></span>h of incubation&#44; 30&#37; was converted into DBT and 20&#37; into MBT and the remaining amount of TBT was converted into inorganic tin&#46; In the case of strain HS5&#44; the mean percentage of TBT that remained was almost 50&#37;&#44; after 40<span class="elsevierStyleHsp" style=""></span>h of incubation&#44; 25&#37; was converted into DBT and 15&#37; into MBT&#46; According to the above results&#44; strain HS1 and HS5 can be classified as moderate TBT degraders&#46; Strain HS2 degraded more than 90&#37; of TBT&#44; converting 50&#37; into DBT and 35&#37; into MBT&#46; The degradation proceeded according to the postulated scheme &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0090" class="elsevierStylePara elsevierViewall">Finally&#44; strain HS2 showed the highest TBT degradation capability&#44; exceeding HS1 and HS5 abilities &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Phylogenetic analysis of 16S rRNA gene for <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41; clearly shows 100&#37; similarity with <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> LPM-5 and 93&#37; similarity with <span class="elsevierStyleItalic">Stenotrophomonas maltophilia</span> KB2&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">The TBT degradation profile over 40<span class="elsevierStyleHsp" style=""></span>h of incubation at 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; pH 9 and salinity 7&#37; &#40;w&#47;v&#41; for strain HS2 is shown in <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B&#46; Between 10 and 30<span class="elsevierStyleHsp" style=""></span>h a decrease in TBT concentration was observed&#44; which was consistent with the rise in bacterial growth&#46; Between 30 and 50<span class="elsevierStyleHsp" style=""></span>h&#44; the degradation rate declined along with the available nutrients&#44; achieving more than 95&#37; of degradation within 50<span class="elsevierStyleHsp" style=""></span>h&#46; The degradation of TBT contrasted with the generation of DBT and MBT as intermediate products&#46; DBT concentration increased during incubation&#44; reaching around 50&#37; of the original TBT concentration &#40;2<span class="elsevierStyleHsp" style=""></span>mM&#41;&#44; and MBT was detected with 35&#37; during this period of incubation&#46; The culture medium showed the brown&#44; yellow and white colors that coincided with the postulated products that could be inorganic tin in the form of insoluble compounds &#40;data not shown&#41;&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Antibiotic susceptibility of <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2</span><p id="par0100" class="elsevierStylePara elsevierViewall">Antibiotic susceptibility tests of strain HS2 showed that it was resistant to ampicillin&#44; amoxicillin-clavulanic acid 30<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;disc as &#946;-lactams antibiotics&#59; however&#44; it was susceptible to all other antibiotics tested&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Detection and expression of a gene involved in tributyltin &#40;TBT&#41; resistance in <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2</span><p id="par0105" class="elsevierStylePara elsevierViewall">In the present study&#44; a gene was amplified using specific primers designed based on the TBT gene resistant sequence from TBT degrading bacterium <span class="elsevierStyleItalic">A&#46; molluscorum</span> Av27<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">7</span></a>&#46; The obtained fragment was cloned in <span class="elsevierStyleItalic">E&#46; coli</span> DH5&#945; using pGEM<span class="elsevierStyleSup">&#174;</span>-T Easy Vector&#46; This clone contained pGEM<span class="elsevierStyleSup">&#174;</span>-T Easy vector with an inserted fragment of &#8764;725<span class="elsevierStyleHsp" style=""></span>bp&#44; whose deduced amino acid sequence has high homology &#40;93&#37; homology&#41; with an aryl-sulfatase from <span class="elsevierStyleItalic">S&#46; maltophilia</span> and &#40;87&#37; homology&#41; with an arsenic efflux pump membrane protein from <span class="elsevierStyleItalic">Xanthomonas oryzea</span> and was identified as TBTB-permease &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><p id="par0110" class="elsevierStylePara elsevierViewall">To verify whether the TBTB permease gene was expressed in response to TBT&#44; RT-PCR experiments were performed with total RNA extracted from <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 growing on TBT and fructose&#46; RT-PCR amplification products of the expected 400-bp size were observed with 3<span class="elsevierStyleHsp" style=""></span>pg of RNA extracted from the culture grown on TBT &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>B&#41;&#44; whereas no product was detected with RNA extracted from the culture grown on fructose &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>A&#41;&#44; indicating that gene transcripts are induced at least 70-fold and also specifically induced in the presence of TBT&#46; In addition&#44; no amplification products were observed in controls devoid of reverse transcriptase or template cDNA &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>B lane C1&#8242; and lane C2&#8242;&#41;&#46; Sequencing of the approximately 400-bp product confirmed that it was identical to the corresponding TBTB permease gene fragment from genomic DNA of <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2&#46; These results showed direct evidence that the TBTB-permease gene product from <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 is definitely involved in TBT resistance&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Discussion</span><p id="par0115" class="elsevierStylePara elsevierViewall">Tributyltin &#40;TBT&#41; is considered a recalcitrant compound used as a biocide in the antifouling paints of boats and ship hulls&#44; and then released slowly to the aquatic ecosystem in considerable amounts with toxic effect to a large number of aquatic organisms<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">3</span></a>&#46; Despite the toxic effect of TBT on eukaryotic or prokaryotic organisms<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">1</span></a>&#44; some microorganisms are resistant to TBT<a class="elsevierStyleCrossRef" href="#bib0350"><span class="elsevierStyleSup">35</span></a>&#46; TBT degradation by the three strains decreased as salinity increased&#59; furthermore&#44; no degradation occurred at salinity greater than 10&#37; NaCl &#40;w&#47;v&#41; even with a long incubation time&#46; This observation is consistent with those noted in extremely halotolerant bacteria<a class="elsevierStyleCrossRef" href="#bib0340"><span class="elsevierStyleSup">33</span></a>&#46; Although much of the research on TBT degradation in terrestrial and marine environments has been studied extensively under oxic conditions<a class="elsevierStyleCrossRefs" href="#bib0210"><span class="elsevierStyleSup">7&#44;24</span></a>&#44; little is known about TBT degradation in hypersaline environments&#46; Bioremediation of polluted hypersaline wastewaters and other environments with nonhalophilic microorganisms is difficult because salt inhibits their growth and the degradation of organic compounds<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">30</span></a>&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">The HS2 strain had higher ability to degrade TBT than HS1 and HS5 and showed 99&#37; similarity with <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> LPM-5&#44; the most closely related organism&#44; which is an aerobic EDTA-degrading bacterium<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">25</span></a> and 93&#37; with <span class="elsevierStyleItalic">S&#46; maltophilia</span> KB2&#44; which is a triphenyltin-degrading bacterium<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">16</span></a>&#46; The HS2 strain has the ability to use TBT as carbon source in a mineral salt medium with 7&#37; salinity and pH 9&#46; Furthermore&#44; TBT degraders <span class="elsevierStyleItalic">A&#46; molluscorum</span> Av27 and <span class="elsevierStyleItalic">Aeromonas veronii</span> Av27&#44; had also the capacity to use TBT as carbon source in a mineral salt medium<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">5</span></a> and showed optimum growth with salinity 4&#37; &#40;w&#47;v&#41; and pH 9<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">6</span></a>&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Antibiotic test results confirm that the HS2 strain does not exhibit potential risk of toxicity and could be used for TBT bioremediation&#46; The resistance of common antibiotics could be toxic and affect human health<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">14</span></a>&#46; Moreover&#44; the rapid appearance of antibiotic resistance among environmental bacteria as a result of horizontal gene transfer through plasmids&#44; transposons and integrons is a known process<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">17</span></a>&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">To our knowledge&#44; this is the first reported halophilic and alkaliphilic bacteria from the Mediterranean Sea that has the ability to degrade TBT under both halophilic and alkaliphilic conditions&#46; The results suggested that <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 degraded TBT into DBT and MBT as less toxic compounds and this haloalkaliphilic strain could be applied in the remediation of TBT contaminants or natural attenuation of extreme alkaline and hypersaline TBT contaminated sites&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">TBT degraders received great attention&#44; especially genes&#44; pathways&#44; and mechanisms of their degradation&#59; however&#44; until now the cellular and molecular mechanisms involved in TBT resistance are not clear and they are supposed to be different either among bacterial genera or even the same species or strain<a class="elsevierStyleCrossRefs" href="#bib0210"><span class="elsevierStyleSup">7&#44;24</span></a>&#46; Furthermore&#44; it is not known whether haloalkaliphilic bacteria degrade TBT using novel genes and pathways compared to those used by non haloalkaliphiles&#46; HS2 harbor the TBTB-permease gene which is a single&#44; non-redundant&#44; protein sequence&#44; where anion permease like ArsB has been shown to export arsenate and antimonite in eubacteria and archaea&#46; A typical ArsB permease contains 8&#8211;13 transmembrane helices and can function as a chemiosmotic transporter or as a channel-forming subunit of an ATP-driven anion pump&#46; ArsB proteins belong to the ArsB&#47;NhaD superfamily of permeases that translocate sodium&#44; arsenate&#44; sulfate&#44; and organic anions across biological membranes<a class="elsevierStyleCrossRef" href="#bib0330"><span class="elsevierStyleSup">31</span></a>&#46; Most metal resistance systems included tin accomplished by ATPase or a single polypeptide like &#40;ArsB&#41; efflux systems<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">10</span></a>&#46; These systems could explain the ability of TBT resistant bacteria&#44; either by formation of DBT and MBT as a result of removing butyl groups from the tin atom<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">5</span></a>&#44; or pumping out TBT through a multidrug efflux pump<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">24</span></a> or TBT bioaccumulation in the bacterial cells<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">11</span></a>&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">This work presented the important role of TBT-degrading bacteria isolated from the Mediterranean Sea at a TBT-contaminated site&#44; where three halophilic and alkaliphilic bacterial strains <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">R&#46; borbori</span> HS5 were isolated&#44; having the ability to use TBT as a carbon source in minimal media with 7&#37; &#40;w&#47;v&#41; salinity and pH 9&#46;</p><p id="par0145" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 was the most efficient TBT degrader and had the ability to transform most of the TBT into DBT and MBT&#46; In TBT-resistant bacterium <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2&#44; a new gene was identified as TBTB-permease&#44; which had 93&#37; similarity with aryl sulfatase in <span class="elsevierStyleItalic">S&#46; maltophilia</span> and 87&#37; similarity with arsenic efflux pump membrane protein in <span class="elsevierStyleItalic">X&#46; oryzea</span>&#46; The TBTB-permease gene was only expressed in the presence of TBT&#46; The three novel haloalkaliphilic strains and their TBT degradation ability have never been reported previously&#46; Moreover&#44; HS2 strain is an effective candidate bacterium for TBT bioremediation in halophilic and alkaliphilic TBT contaminated sites&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Conflict of interest</span><p id="par0150" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
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        0 => array:3 [
          "identificador" => "xres1167518"
          "titulo" => "Abstract"
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              "identificador" => "abst0005"
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        1 => array:2 [
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          "titulo" => "Resumen"
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        3 => array:2 [
          "identificador" => "xpalclavsec1092323"
          "titulo" => "Palabras clave"
        ]
        4 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
        ]
        5 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:8 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Isolation and identification of haloalkaliphilic bacteria degrading TBT"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "TBT sensitivity"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Antibiotic susceptibility"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Effect of pH and salinity on TBT degrading ability"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Growth curve and TBT analysis"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Amplification of TBT degrading gene"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Extraction of mRNA&#44; cDNA synthesis&#44; and RT-PCR"
            ]
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              "identificador" => "sec0050"
              "titulo" => "Nucleotide sequence accession number"
            ]
          ]
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        6 => array:3 [
          "identificador" => "sec0055"
          "titulo" => "Results"
          "secciones" => array:4 [
            0 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Haloalkaliphilic bacteria isolation and identification"
            ]
            1 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "TBT sensitivity and degradation by the three identified haloalkaliphilic bacterial strains"
            ]
            2 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Antibiotic susceptibility of S&#46; chelatiphaga HS2"
            ]
            3 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Detection and expression of a gene involved in tributyltin &#40;TBT&#41; resistance in S&#46; chelatiphaga HS2"
            ]
          ]
        ]
        7 => array:2 [
          "identificador" => "sec0080"
          "titulo" => "Discussion"
        ]
        8 => array:2 [
          "identificador" => "sec0085"
          "titulo" => "Conflict of interest"
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        9 => array:1 [
          "titulo" => "References"
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      ]
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    "fechaRecibido" => "2017-08-03"
    "fechaAceptado" => "2017-12-11"
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        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1092322"
          "palabras" => array:4 [
            0 => "Tributyltin"
            1 => "Biodegradation"
            2 => "Haloalkaliphilic"
            3 => "ArsB permease"
          ]
        ]
      ]
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        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Palabras clave"
          "identificador" => "xpalclavsec1092323"
          "palabras" => array:4 [
            0 => "Tributiltina"
            1 => "Biodegradaci&#243;n"
            2 => "Haloalcalif&#237;lico"
            3 => "ArsB permeasa"
          ]
        ]
      ]
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      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Tributyltin &#40;TBT&#41; is recognized as a major environmental problem at a global scale&#46; Haloalkaliphilic tributyltin &#40;TBT&#41;-degrading bacteria may be a key factor in the remediation of TBT polluted sites&#46; In this work&#44; three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea&#46; After analysis of the 16S rRNA gene sequences the isolates were identified as <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">Rhizobium borbori</span> HS5&#46; The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7&#37; &#40;w&#47;v&#41; salt concentration&#46; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin &#40;DBT and MBT&#41;&#46; A gene was amplified from strain HS2 and identified as TBTB-permease-like&#44; that encodes an ArsB-permease&#46; A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance&#46; The three novel haloalkaliphilic TBT degraders have never been reported previously&#46;</p></span>"
      ]
      "es" => array:2 [
        "titulo" => "Resumen"
        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Se considera a la tributiltina &#40;TBT&#41; como un problema medioambiental serio a escala global&#46; Las bacterias haloalcalif&#237;licas degradadoras de TBT pueden constituir un factor clave para remediar &#225;reas contaminadas con dicho xenobi&#243;tico&#46; En este estudio se aislaron 3 cepas de bacterias haloalcalif&#237;licas procedentes de un sitio contaminado con TBT en el mar Mediterr&#225;neo&#46; Tras analizar las secuencias del gen de 16S del ARNr&#44; se identificaron los aislados como <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span> HS2 y <span class="elsevierStyleItalic">Rhizobium borbori</span> HS5&#46; Las condiciones de crecimiento &#243;ptimas para la biodegradaci&#243;n de TBT por parte de las 3 cepas fueron pH 9 y 7&#37; &#40;p&#47;v&#41; de concentraci&#243;n de sal&#46; <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 fue el degradador de TBT m&#225;s efectivo&#44; con capacidad de transformar la mayor parte de ese compuesto en dibutiltina y monobutiltina &#40;DBT y MBT&#41;&#46; Se amplific&#243; un gen de la cepa HS2&#44; que fue identificado como tipo TBTB-permeasa&#44; que codifica para una ArsB permeasa&#46; Un an&#225;lisis de la cepa HS2 por reacci&#243;n en cadena de la polimerasa con transcriptasa inversa &#40;RT PCR&#41; confirm&#243; que el gen TBTB-permeasa contribuye a la resistencia al TBT&#46; Estos 3 nuevos degradadores haloalcalif&#237;licos de TBT no hab&#237;an sido reportados previamente&#46;</p></span>"
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          "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Proposed TBT debutylation pathway based on this study and other studies<a class="elsevierStyleCrossRefs" href="#bib0305"><span class="elsevierStyleSup">26&#44;35</span></a>&#46;</p>"
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          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Growth of <span class="elsevierStyleItalic">Sphingobium</span> sp&#46; HS1&#44; <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span> HS2 and <span class="elsevierStyleItalic">Rhizobium borbori</span> HS5 on 2<span class="elsevierStyleHsp" style=""></span>mM TBT as a carbon source&#46; Growth was monitored by following colony-forming units &#40;CFU&#41;&#44; TBT depletion and formation of DBT and MBT were assessed by GC&#46;</p>"
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          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Phylogenetic tree based on 16S rRNA gene sequences showing the relationship with <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span>&#46; HS2 strain &#40;accession number KR780649&#41; pointed by an arrow to other reported <span class="elsevierStyleItalic">Stenotrophomonas</span> strains&#46; The strain followed by the accession numbers and degraded substrate&#59; petroleum hydrocarbon &#40;petroleum H&#41;&#44; benzene &#40;BTEX&#41;&#44; toluene&#44; ethylbenzene and xylenes&#44; triphenyltin &#40;TPT&#41;&#44; ethylenediaminetetra acetic acid &#40;EDTA&#41;&#44; and tributyltin &#40;TBT&#41; degraders&#46;</p>"
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          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Phylogenetic tree showing the relatedness on the basis of TBTB permease&#46; The dendrogram was calculated using Phylogeny&#46;fr based on protein sequence alignments&#46; TBTB permease proteins from <span class="elsevierStyleItalic">Stenotrophomonas chelatiphaga</span> HS2 are pointed by an arrow&#46; The scale bar corresponds to an estimated evolutionary distance of 1 amino acid substitution per site&#46;</p>"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">RT-PCR amplification of TBTB permease mRNA from <span class="elsevierStyleItalic">S&#46; chelatiphaga</span> HS2 grown on fructose &#40;A&#41; or TBT &#40;B&#41;&#46; M lanes contain the molecular size marker Hyperladder 1 &#40;Bioline&#41;&#46; cDNA generated from 1<span class="elsevierStyleHsp" style=""></span>&#956;g template RNA was serially diluted &#40;3&#46;2-fold&#41; with nuclease-free water&#44; and 1<span class="elsevierStyleHsp" style=""></span>&#956;l of each dilution was subjected to amplification by PCR &#40;lanes 1 to 6&#41; for cDNA from fructose and &#40;lanes 1&#8242; to 5&#8242;&#41; for cDNA from TBT&#46; Negative controls included undiluted RT-PCR mixtures devoid of reverse transcriptase &#40;lane C1&#8242;&#41; or template cDNA &#40;lane C2&#8242;&#41;&#46; PCR mixtures containing 1<span class="elsevierStyleHsp" style=""></span>ng of genomic DNA as a template were used as positive control &#40;lane C&#41; in the same experiment&#46;</p>"
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      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0015"
          "bibliografiaReferencia" => array:35 [
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Article information

ISSN: 03257541
Original language: English

DOI:10.1016/j.ram.2017.12.002

The statistics are updated each day

Year/Month	Html	Pdf	Total

2024 October	7	3	10
2024 September	26	3	29
2024 August	21	8	29
2024 July	15	3	18
2024 June	16	1	17
2024 May	16	6	22
2024 April	20	6	26
2024 March	27	5	32
2024 February	26	1	27
2024 January	24	5	29
2023 December	14	8	22
2023 November	24	2	26
2023 October	42	12	54
2023 September	31	3	34
2023 August	21	2	23
2023 July	20	4	24
2023 June	17	4	21
2023 May	65	3	68
2023 April	38	5	43
2023 March	40	8	48
2023 February	29	3	32
2023 January	20	3	23
2022 December	24	4	28
2022 November	25	13	38
2022 October	21	9	30
2022 September	16	11	27
2022 August	26	7	33
2022 July	19	6	25
2022 June	35	4	39
2022 May	66	10	76
2022 April	64	8	72
2022 March	125	17	142
2022 February	144	4	148
2022 January	102	13	115
2021 December	50	19	69
2021 November	82	9	91
2021 October	57	12	69
2021 September	41	12	53
2021 August	26	5	31
2021 July	25	10	35
2021 June	18	8	26
2021 May	32	13	45
2021 April	56	41	97
2021 March	33	17	50
2021 February	16	15	31
2021 January	16	19	35
2020 December	12	5	17
2020 November	15	10	25
2020 October	12	7	19
2020 September	19	8	27
2020 August	17	14	31
2020 July	22	11	33
2020 June	18	7	25
2020 May	13	11	24
2020 April	12	4	16
2020 March	22	3	25
2020 February	19	6	25
2020 January	14	7	21
2019 December	29	5	34
2019 November	21	4	25
2019 October	26	6	32
2019 September	29	9	38
2019 August	17	3	20
2019 July	15	2	17
2019 June	38	27	65
2019 May	72	26	98
2019 April	48	14	62
2019 March	10	14	24
2019 February	2	47	49
2019 January	2	2	4
2018 December	1	4	5
2018 November	3	4	7
2018 October	8	18	26
2018 September	4	5	9
2018 August	0	4	4
2018 July	1	5	6
2018 June	2	9	11
2018 May	0	7	7
2018 April	0	17	17

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