Pretravel assessment of VFR travelers can be enhanced by addressing specific topics within the domains of the determinants of health listed in Table 2. Clinicians can use this approach to identify specific gradients of risk for VFR travelers in multiple areas in addition to infectious diseases. A more nuanced approach is also possible for travelers who may appear very different but in fact have quite similar risk profiles, or Ion Channel Ligand Library molecular weight who appear similar but in fact may have quite different risks. Risk assessment within these additional domains also encourages increased attention

to factors and outcomes other than infectious diseases, such as road traffic accidents, air pollution, personal safety, psychological and psychosocial issues, and exposures to extremes of climate or severe weather events. This framework for risk assessment can also be applied to urban-rural migration within a country (such as see more moving from an urban area of Brazil into a yellow fever endemic area, or moving, in many countries, from a relatively

safe rural area into a large urban area with risks of urban violence, poorer sanitation, and air pollution). As inter-regional travel increases and classic travel risks move away from infectious disease risks to a broader concept of travel-related health problems,21 it will be necessary to explore in more depth the risk gradient for VFR travelers in these different domains. Application of this framework for VFR travelers will be new to many clinicians, tetracosactide though most travel medicine practitioners are already familiar with the process of risk assessment that is used in the routine practice of travel medicine. To facilitate use of the new definition specific to VFR travelers, case scenarios have been developed that illustrate application of the definition.22 These cases will assist clinicians in understanding the difficulties incurred when

using legal status or ethnicity to determine risk. Over time, this framework should facilitate design of studies involving VFR travelers. Global security and migration-related illness are topics of increasing international importance.23,24 Acknowledging the increased role of VFR travel and potential for transmission of infectious diseases has been seen with respect to influenza, HIV infection, tuberculosis, hepatitis A, dengue, chikungunya, malaria, and other infectious diseases.25,26 Noninfectious causes of morbidity may include exposure to counterfeit or adulterated medications,27,28 contaminated or poisonous foods (melamine-contaminated dairy products), accidents, physical or sexual violence, and exposure to air pollution or high altitude. Examples of public health initiatives to address potentially travel-related noninfectious disease issues include “Look Right” signs in the UK and education and efforts to improve air quality around the time of the Beijing 2008 Olympics.

, 1999). BIME-1 and BIME-2 correspond to SMAG TT and HH dimers. However, HH dimers are about 10 times more abundant than TT dimers. In contrast, BIME-1 (74 repeats) are three times more abundant than BIME-2 (24 repeats).

Moreover, both BIME-1 and BIME-2 are invariably comprised of elements from different subfamilies (Bachellier et al., 1999; see also http://www.pasteur.fr/recherche/unites/pmtg/repet/index.html). The predominance Akt inhibitor of TT over HH dimers, and the composite nature of dimers, is also a distinctive feature of the abundant REP families found in Pseudomonas putida (Aranda-Olmedo et al., 2002) and P. syringae (Feil et al., 2005). It has been hypothesized that REPs are mobilized by a mTOR inhibitor transposase of the IS200/IS605 family, and the corresponding genes have been shown to be flanked by REPs in many species (Nunvar et al., 2010). Four genes encoding this transposase were identified in K279a DNA (ORFs 1101,

1152, 2816 and 4509), but only ORFs 1101 and 2816 are flanked by SMAGs. We believe that REPs are an ancient component of the genomes of Proteobacteria, which have been actively mobilized by transposition only early in their history. According to this view, REPs disappeared in time from most species, their dissemination being plausibly detrimental to the cell, and have been maintained only in species in which they could no longer transpose. This hypothesis is supported by the observation that SMAG sequences were found in none of the 41 species-specific GEIs, plausibly acquired by lateral gene transfer, which account for >10% of the K279a chromosome (Rocco et al., 2009). REPs are similarly restricted to core genome regions in P. syringae (Tobes & Pareja, 2005). In contrast to what was observed for REPs in other species (Tobes & Pareja, 2006), SMAGs are not targeted by mobile DNA. However, it is worth noting that a K279a GEI encoding type 1 pili (Rocco et al., 2009) is flanked by SMAG-2 dimers. L-NAME HCl About 1/7 of the ORFs of the K279a strain are flanked by SMAGs in a distance range that makes the presence of promoter or terminator

sequences unlikely. It is plausible that most of these elements are transcribed into mRNA, and that their folding into RNA hairpins may influence the level of expression of flanking genes. The number of genes potentially controlled at the post-transcriptional level by SMAGs may be higher than estimated, because many repeats are inserted either upstream (17 elements) or downstream (150 elements) or within (44 elements) known or putative operons. We analyzed genes transcribed in the same direction intermingled with SMAG sequences, and found that the repeats influence the segmental mRNA stability. Both monomers and dimers function as stabilizers of upstream transcripts, and work with comparable efficiency when embedded in the same RNA context (Fig. 5).

3b). This contrasted with the finding in Pseudomonas aeruginosa PAO1, a wound isolate (Stover et al., 2000), that the expression of the anthranilate dioxygenase operon was strongly dependent on iron (Oglesby et al., 2008). This difference might be owing to different habitats to which the two strains have been adapted. Pseudomonas aeruginosa PAO1 might have acquired selleck inhibitor a regulatory system that stringently responds to external iron conditions, that is, strictly down-regulates the anthranilate dioxygenase gene in animal infections, where the iron resource is severely limited. The

ATCC 17616, which has been living in soil where iron is not so severely limited, might have developed a regulatory system that does not tightly control the expression of genes for iron-requiring enzymes. The reason for the higher activity of andA promoter in the fur mutant when 2,2′-dipyridil was present (Fig. 3b) is not clear. However, our recent findings suggested a higher level of ferric ion in the fur mutant, leading to the generation of a higher level of hydroxyl radical by Fenton reaction, which might have adverse effects on the promoter activity. The addition of 2,2′-dipyridil might have alleviated such

effects. In this regard, the decreased promoter activity of the fur mutant might be the combined effects of the increased hydroxyl radical and the transcriptional regulations that were directly or indirectly mediated by Fur. When grown in 1/3-LB medium, ATCC 17616 cells required more than 50 μM of anthranilate for the induction tuclazepam of the andA promoter (data not shown). The concentration of anthranilate in the soil extract prepared by ethyl acetate was below the detection limit of our experimental GSK-3 inhibitor review devices (Nishiyama et al., 2010), which could be around 0.1 μM (data not shown). In addition, the andA promoter activity was low during

the initial colonization period and only increased after 4 days in the soil environment, indicating that the inducer is not present during the first few days of colonization (Fig. 4). Therefore, a simple explanation that anthranilate present in the soil sample induced the andA operon seems to be unlikely. During the initial period of colonization in the soil, the cellular concentration of anthranilate or tryptophan might have increased to a level sufficient to induce the andA operon. There are several possible sources of anthranilate or tryptophan. One possible source is proteins that were present in the cells being inoculated. At the beginning of the incubation in the soil, the cellular proteins might have been used as the resources to change cellular physiological status to fit the soil environment. In such a case, tryptophan might accumulate and trigger anthranilate catabolism. As tryptophan and anthranilate are not good growth substrates, their catabolism might be of low priority and therefore might tend to accumulate in the cells. Other possible source is proteins and metabolites released into the environment from lysed cells.

coelicolor (Yang et al., 2006). The specificity for dCMP incorporation into pORF102 leaves the possibility that either the

Selleckchem Ponatinib first or the second nucleotide of the 3′-end of pAL1 (… GCAGG-3′) may serve as a template for the deoxynucleotidylation reaction. In this study, we identified the gene product of pAL1.102 as a protein that is associated with both termini of the linear Arthrobacter plasmid pAL1. The proposed TP – at least when fused to MBP to ensure solubility – was not capable of specifically recognizing telomeric pAL1 DNA in vitro. However, in an in vitro deoxynucleotidylation assay, the pORF102 protein specifically incorporated dCMP, complementary to the 3′-ends of pAL1. This is consistent with its presumed role as a protein primer in DNA replication. The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged (FE 383/11). We thank Prof. Dr R. Brandsch (University of Freiburg, Germany) for kindly providing the vector pART2, and Prof. Dr A. Steinbüchel (Münster) for access to the phosphoimager. We also thank Manuel Tomm for initial EMSA experiments, and Gabriele Niester and Almut Kappius for technical assistance. Table S1. Primers and ssDNA template

Nutlin-3a mouse used in this study. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Many bacteria produce siderophores for sequestration of growth-essential iron. Analysis of the Salinispora genomes suggests that these

marine actinomycetes support multiple hydroxamate- and phenolate-type siderophore pathways. We isolated and characterized desferrioxamines (DFOs) B and E from all three recognized Salinispora species and linked their biosyntheses in S. tropica CNB-440 and S. arenicola CNS-205 to the des locus through PCR-directed mutagenesis. Gene inactivation of the predicted iron-chelator N-acetylglucosamine-1-phosphate transferase biosynthetic loci sid2-4 did not abolish siderophore chemistry. Additionally, these pathways could not restore the native growth characteristics of the des mutants in iron-limited media, although differential iron-dependent regulation was observed for the yersiniabactin-like sid2 pathway. Consequently, this study indicates that DFOs are the primary siderophores in laboratory cultures of Salinispora. Siderophores are small molecules secreted by bacteria to sequester growth-essential ferric iron that is poorly soluble under neutral pH and aerobic conditions (Neilands, 1995). The structures of siderophores vary considerably and are often suited to the environmental niche of the producing bacterium. For example, amphiphilic siderophores possess hydrophobic fatty acid chains that enable them to remain associated with the cell membrane (Martinez et al., 2003) – an attribute particularly advantageous in pelagic marine environments where dilution occurs rapidly.

Each growth condition was repeated once. Total RNA was extracted according to the protocol provided by Qiagen (RNeasy Mini Kit). For cell harvest, 2 volumes of RNAprotect Bacteria Reagent (Qiagen) were added to 1 volume bacterial culture and mixed vigorously. The solution was incubated at room temperature for 5 min and immediately centrifuged at 5000 g for 10 min. For cell lysis, the cell pellet was resuspended in a 10% aliquot of the initial

Dabrafenib chemical structure sample volume containing 1 mg mL−1 lysozyme in 10 mM Tris/HCl, 1 mM EDTA, pH 8.0, and incubated at room temperature for 20 min. Then, 1.8 mL RLT buffer (Qiagen) containing 1% (v/v) β-mercaptoethanol was added and mixed intensively, Omipalisib followed by the addition of 1.2 mL ethanol.

The RNA solution was purified using the RNeasy Mini Kit, by applying the total volume stepwise to one column. On-column DNase digestion was performed twice for 20 min to ensure the complete removal of genomic DNA. RNA integrity and purity were checked by agarose gel electrophoresis. cDNA synthesis was performed from about 10 μg total RNA with a statistically distributed mixture of hexanucleotides as primers (random priming) using SuperscriptII (Invitrogen) reverse transcriptase according to the manufacturer’s protocols. An aliquot of 25 μg cDNA was sequenced using the Genome Analyzer II at GATC Biotech AG (Konstanz). For this, the cDNA was nebulized to generate fragments <800 bp long. A terminal ‘A’ was then transferred to the 3′ end and cDNA fragments were ligated to adapters, purified and bridge amplified. Thirty-six cycles of sequencing-by-synthesis were performed 3-oxoacyl-(acyl-carrier-protein) reductase for each library using the Genome Analyzer GAII SR. illumina genome analyzer pipeline

software (version 0.2) was used to qualify reads (Klockgether et al., 2010). Sequence reads that passed the default signal quality filter and were not aligned by ELAND (Efficient Large-Scale Alignment of Nucleotide Databases) to a reference of the P. putida rRNA genes were used for gene expression analysis. The reads were subsequently aligned to the P. putida genome (NC_002947.3) using the bowtie software package (Langmead et al., 2009). The remaining reads mapped to rRNA were subsequently excluded with a custom PERL script. Four nucleotides were trimmed from the 3′ end of each read and a seed size of 28 bp was used, in which two mismatches were allowed. The quality mismatch sum was 100 and results were transformed into a SAM format (command line: bowtie -t putida -l 28 -e 100 –best –sam -3 4 -n 2 -p 7). A summary table was then generated using the integrative web analysis tool galaxy (Giardine et al., 2005). The functions ‘coverage’ and ‘join’ were used, respectively, to summarize (1) the coverage of each ORF from the P. putida NCBI annotation (version NC_002947.

All the mutants obtained in this study exhibited significantly decreased susceptibility Ku-0059436 in vivo to lincomycin (MICs ≥512 μg mL−1), chloramphenicol (MICs ≥64 μg mL−1) and florfenicol (MICs ≥512 μg mL−1), and three mutants (mutants PV10, ST7 and SV10) showed cross-resistance to erythromycin (MICs ≥256 μg mL−1), tilmicosin (MICs ≥256 μg mL−1) and tylosin (MICs ≥16 μg mL−1). The three subcultured clones were analyzed by amplification and sequencing of the domain V of 23S rRNA gene and ribosome protein L3. Nucleotide

sequences were always identical for the three clones. As mutations in ribosome protein L3 are responsible for decreased pleuromutilin susceptibility in several bacteria species (Bøsling et al., 2003; Pringle et al., 2004; Kosowska-Shick et al., 2006; Gentry et al., 2007), we first examined the sequences of ribosome protein L3 for the mutants selected in this study. None of these mutants were found to possess ribosome www.selleckchem.com/products/LBH-589.html protein L3 mutations. Several mutations were found in domain V of 23S rRNA gene. Although M. gallisepticum contains two copies of the 23S rRNA gene, mutations were always present in only one of the two 23S rRNA gene

alleles (Table 2). All the mutants with decreased susceptibility to tiamulin and valnemulin possessed the A2503U mutation in 23S rRNA gene. Of these mutants, four (mutants PT3, ST3, PV4 and SV4) harbored the A2503U mutation in 23S rRNA gene and did not have any other alterations. The MICs of tiamulin (MICs=0.5–1 μg mL−1) and valnemulin (MICs=0.032–0.063 μg mL−1) for these mutants showed a significant increase in comparison with those for the parental strains. Combinations of two or three mutations were selected in this study. Mutant PT10 possessed the A2503U mutation and an additional G2061U mutation in 23S rRNA gene. The MICs of tiamulin and valnemulin Megestrol Acetate for this mutant increased to 8 and 0.25 μg mL−1, respectively.

Mutants ST10 and SV7 possessed the A2503U mutation and an additional G2447A mutation. For both mutants, this combination of two mutations led to an increase in the MICs of tiamulin and valnemulin to 32 and 8 μg mL−1, respectively. In addition to the A2503U mutation, an A2058G mutation and an A2059G mutation were found in mutants PV10 and ST7, respectively. Both mutants exhibited significantly decreased susceptibility not only to tiamulin and valnemulin but also to macrolide antibiotics erythromycin, tylosin and tilmicosin (Table 2). The latter cross-resistance phenotype may be due to the presence of the A2058G mutation (mutant PV10) and the A2059G mutation (mutant ST7).

“
“Serotonin (5-HT) Ibrutinib molecular weight signaling in the central nervous system (CNS) helps to regulate a variety of important cognitive and behavioral processes and it is a common therapeutic target for mood disorders. Because sleep abnormalities are frequently associated

with mood disorders, there has been substantial interest in the regulatory abilities of 5-HT signaling on the sleep/wake cycle. However, to date there have been few practical and reliable ways to reversibly manipulate brain 5-HT levels without disrupting other monoaminergic signaling pathways that may be important for sleep. In this issue of European Journal of Neuroscience, Nakamaru-Ogiso and colleagues reveal a new method for reducing brain 5-HT levels in rats, a well-established MAPK inhibitor rodent model of sleep–wake architecture. Intraperitoneal injections of the hemoprotein enzyme tryptophan side chain oxidase I (TSOI) transiently reduce brain and peripheral 5-HT concentrations by reversibly depleting the rats of tryptophan, while preserving catecholeaminergic

signaling. The authors report that this transient reduction of brain 5-HT abolishes the sleep/wake rhythm but has no meaningful influences on daily sleep amount. Moreover, the circadian rhythm in brain temperature is preserved in TSOI-injected rats, providing evidence that the effects of the manipulation are specific to sleep and are not caused by global effects on circadian timing. These findings suggest that in addition to its well-established D-malate dehydrogenase regulatory influences

on central circadian timing, brain 5-HT also plays a more direct role in the specific regulation of the sleep/wake rhythm. The lack of practical methods to rapidly and reversibly manipulate brain 5-HT in mammals has been an obstacle in our understanding of the role of 5-HT signaling in sleep. Tryptophan-hydroxylase 2 (TPH2), the rate-limiting enzyme in 5-HT synthesis in the brain, has been a dependable target for brain 5-HT reduction; however, a lack of specificity of TPH2 inhibitors results in the collateral reduction of catecholamines such as the sleep/wake regulator norepinephrine, making these types of agents impractical for sleep studies. Serotonergic neurotoxins and TPH2 molecular deletions in mice have also been valuable to uncover the specific roles of 5-HT signaling, but neither manipulation is reversible, giving them limited usefulness in in vivo sleep experiments. Nakamaru-Ogiso and colleagues report that TSOI eliminates tryptophan and reduces brain 5-HT levels to 30% of controls within 12 h of treatment, with no collateral reductions in catecholeamines, other amino acids or protein synthesis. These influences of TSOI injection are no longer observed 96 h after injection.

The clinical and virological characteristics of the 76 genotype 1 HIV/HCV-coinfected patients are presented in Table 1. Patients’ characteristics did not differ between the group of six HIV/HCV-coinfected

patients harbouring HCV protease mutations and those without known HCV PI resistance mutations (Table 2). All of the sequences from HIV/HCV genotype 4-coinfected patients and those retrieved from the GenBank database had amino acid changes at position 36 (V36L) shown to confer decreased susceptibility to telaprevir. Finally, the NS3 catalytic triad (H57, D81 and S139) was highly conserved among the 120 sequences from HIV/HCV-coinfected KU-60019 mw patients. We found no significant difference in natural polymorphisms at positions associated with

HCV PI resistance between HCV-monoinfected and HIV/HCV-coinfected patients. Our results are in accordance with those of a study by Halfon et al. in a small group of patients. They did not find any difference in observed mutation rates between HCV-monoinfected and HIV/HCV-coinfected patients (19% and 18%, respectively) at positions associated with HCV PI resistance [9]. In contrast, Morsica et al. found a higher prevalence of HCV PI resistance mutations in 37 sequences obtained from coinfected patients in comparison with 250 sequences from HCV-monoinfected patients retrieved from the GenBank database (16.2% and 0.8%, respectively) [8]. In our study, which included a large number of patients, previous HCV treatment did not seem to influence the prevalence of HCV PI resistance mutations. No patient showed substitutions

at position A156, which are known to confer the highest level of resistance Galunisertib nmr to telaprevir or boceprevir. The role of other mutations is difficult to predict, but the possibility that they may have an impact on the virological response to treatment cannot be excluded and needs to be investigated. Indeed, HCV strains with naturally occurring mutations that may confer resistance to HCV PIs show reduced fitness and are generally sensitive to interferon and/or interferon plus ribavirin therapy regimens. The role of these mutations in long-term therapy Metalloexopeptidase and the likelihood of viral breakthroughs are still to be determined, in particular in patients who are nonresponders to previous interferon-based therapy or relapsers on this therapy. The preservation of the NS3 catalytic triad, as observed in our study, is probably attributable to functional constraints on the protease. Its structural and chemical integrity is required to process the HCV polyprotein. All sequences from genotype 4-infected patients contained mutation V36L, which is known to confer decreased susceptibility to telaprevir [11]. Large clinical trials to better document the efficacy of STAT-C in patients infected with genotype 4 are required. Our study on sequences from 120 HIV/HCV-coinfected patients suggests that the natural prevalence of strains resistant to HCV PIs does not differ between HCV-monoinfected and HIV/HCV-coinfected patients.

The clinical and virological characteristics of the 76 genotype 1 HIV/HCV-coinfected patients are presented in Table 1. Patients’ characteristics did not differ between the group of six HIV/HCV-coinfected

patients harbouring HCV protease mutations and those without known HCV PI resistance mutations (Table 2). All of the sequences from HIV/HCV genotype 4-coinfected patients and those retrieved from the GenBank database had amino acid changes at position 36 (V36L) shown to confer decreased susceptibility to telaprevir. Finally, the NS3 catalytic triad (H57, D81 and S139) was highly conserved among the 120 sequences from HIV/HCV-coinfected Selleckchem Ku0059436 patients. We found no significant difference in natural polymorphisms at positions associated with

HCV PI resistance between HCV-monoinfected and HIV/HCV-coinfected patients. Our results are in accordance with those of a study by Halfon et al. in a small group of patients. They did not find any difference in observed mutation rates between HCV-monoinfected and HIV/HCV-coinfected patients (19% and 18%, respectively) at positions associated with HCV PI resistance [9]. In contrast, Morsica et al. found a higher prevalence of HCV PI resistance mutations in 37 sequences obtained from coinfected patients in comparison with 250 sequences from HCV-monoinfected patients retrieved from the GenBank database (16.2% and 0.8%, respectively) [8]. In our study, which included a large number of patients, previous HCV treatment did not seem to influence the prevalence of HCV PI resistance mutations. No patient showed substitutions

at position A156, which are known to confer the highest level of resistance click here to telaprevir or boceprevir. The role of other mutations is difficult to predict, but the possibility that they may have an impact on the virological response to treatment cannot be excluded and needs to be investigated. Indeed, HCV strains with naturally occurring mutations that may confer resistance to HCV PIs show reduced fitness and are generally sensitive to interferon and/or interferon plus ribavirin therapy regimens. The role of these mutations in long-term therapy Rebamipide and the likelihood of viral breakthroughs are still to be determined, in particular in patients who are nonresponders to previous interferon-based therapy or relapsers on this therapy. The preservation of the NS3 catalytic triad, as observed in our study, is probably attributable to functional constraints on the protease. Its structural and chemical integrity is required to process the HCV polyprotein. All sequences from genotype 4-infected patients contained mutation V36L, which is known to confer decreased susceptibility to telaprevir [11]. Large clinical trials to better document the efficacy of STAT-C in patients infected with genotype 4 are required. Our study on sequences from 120 HIV/HCV-coinfected patients suggests that the natural prevalence of strains resistant to HCV PIs does not differ between HCV-monoinfected and HIV/HCV-coinfected patients.

The effects of temperature (20, 25,

30, 37 and 42 °C), initial FE concentration (25, 50, 100 and 200 mg L−1), inoculum size (0.2%, 1%, 5% and 10%), and media pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) on biodegradation of FE were studied. All experiments were performed in triplicate. The degradation of other AOPP herbicides (haloxyfop-R-methyl, screening assay quizalofop-p-ethyl, cyhalofop-butyl and clodinafop-propargyl) by strain T1 was studied under the same conditions. Cells of strain T1 in 100 mL of LB medium grown to stationary phase were harvested by centrifugation (6000 g, 10 min) at 4 °C, washed twice with PBS (50 mM, pH 7.0), and resuspended with 5 mL of PBS. The cells then were disrupted by sonication and the cell debris removed by centrifugation (12 000 g, 20 min) at 4 °C. The supernatant was used as the crude cell-free extract (CFE) for enzyme assay. The assay mixture contained 50 μL of CFE, 3.94 mL of PBS, and 10 μL of FE stock solution to attain a final concentration of 25 mg L−1 FE. The reaction mixture was incubated at 37 °C for 10 min. The concentration of FE was measured

by HPLC following the protocols described below. The negative controls were prepared by STA-9090 in vitro adding 50 μL of CFE, boiled for 30 min, to the assay mixture. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the CFE was performed on a 12% polyacrylamide gel using the Laemmli buffer system (Laemmli, 1970) in the absence of sodium dodecyl sulphate (SDS). After electrophoresis, the gel was cut into two parts. One part was used to analyse the zymogram of esterases by α-napthyl acetate-fast blue B staining (Zhou et al., 2004). The other part was used to

detect the hydrolytic activity of FE by putting the gel on MSM agar plate, which contained 200 mg L−1 FE. After inoculation at 37 °C for 1 h, esterase capable of hydrolysing FE to FA formed a transparent halo. To clone the FE hydrolase gene from Rhodococcus for sp. T1, genome library was constructed by shotgun-cloning technique. The genomic DNA of the bacteria was extracted using the high-salt-concentration precipitation method (Miller et al., 1988). Sau3AI was employed to obtain randomly digested chromosomal fragments. The 2–4.5 kb fragments were recovered using a cleanup kit (Takara Biotechnology Co. Ltd, Dalian, China) and ligated into pUC118(BamHI/BAP). The ligation products were transformed into E. coli DH5α competent cells. The transformants were then spread on LB agar plates containing 100 mg L−1 ampicillin and 200 mg L−1 FE as indicator and incubated at 37 °C for 12–16 h. Positive clones that produced transparent halos were picked from the gene library. The positive recombinant plasmids were extracted and submitted for sequencing by using pUC118(BamHI/BAP) plasmid vector specific primers, subsequently, internal primers were made based on the sequence of the clones and further sequencing analyses were carried out.