Coli DH5alpha Research Articles

Multi-spectroscopic and thermodynamic profiles on HSA binding of Cassia fistula leaf based potential antibacterial and anticancer silver nanoparticles

Here, a simple, one step, lucrative and green synthesis of Cassia fistula leaf extract inspired antibacterial silver nanoparticles (CF-SNPs) was provided. Characterization of these CF-SNPs were achieved by using various spectroscopic techniques for instance Ultraviolet Visible (UV-Vis) Spectroscopy, Fourier-Transform Infrared (FTIR) Spectroscopy, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray (EDX). The effective antibacterial action of the CF-SNPs was checked against Escherichia coli (E. Coli) DH5-Alpha where MIC was 1.6 nM. Anticancer dynamism of the CF-SNPs was also tested in opposition to skin melanoma, A375 cell lines in which 4.4 nM was IC50. The binding proneness of HSA towards CF-SNPs was investigated by means of UV-Vis Spectroscopy, Fluorescence Spectroscopy, Time Resolved Fluorescence Spectroscopy, Circular Dichroism (CD) Spectroscopy, Dynamic Light Scattering, and Isothermal Titration Colorimetry (ITC). CD spectroscopy established minor secondary structural exchange of HSA in HSA-CF-SNPs complex. ITC and Time Resolved Fluorescence Spectroscopy verified the static type quenching mechanism involved in HSA-CF-SNPs complex. The binding constant was 3.45 × 108 M−1 at 298.15K from ITC study. The thermodynamic parameters showed that the interaction was occurred spontaneously by the hydrophilic forces and hydrogen bonding. Communicated by Ramaswamy H. Sarma

An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

Genetic diversity of chromosomal metallo-β-lactamase genes in clinical isolates of Elizabethkingia meningoseptica from Korea

This study was performed to characterize the chromosomal metallo-beta-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5 alpha. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla (GOB) genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla (GOB) gene, including 10 novel variants (bla (GOB-8) to bla (GOB-17)). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5 alpha. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

Salmonella Induces Flagellin- and MyD88-Dependent Migration of Bacteria-Capturing Dendritic Cells Into the Gut Lumen

Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-DeltaSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5alpha by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (DeltafliC DeltafljB)- and SPI1-SPI2 (DeltaSPI1 DeltassrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. Salmonella DeltaSPI1 induced migration of CD11c(+)CX(3)CR1(+)MHCII(+)CD11b(-)CD8alpha(-) DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5alpha or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.

A new serine protease gene from Trichoderma harzianum is expressed in Saccharomyces cerevisiae

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to biocontrol processes. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these proteases for industrial application. We report here a successful expression of the serine protease SL41 from a biocontrol fungus Trichoderma harzianum in Saccharomyces cerevisiae. A new serine proteases gene SL41 has been cloned from T. harzianum. The full length cDNA was isolated by 5' and 3'rapid amplification of cDNA ends. The isolated cDNA of SL41 was then sequenced. The results showed that the open reading frame of SL41 was 1.617 bp long, encoding 538 amino acids. The cloning vector pMD18-T and an E. coli DH5alpha host were used to yield clones as E. coli DH5alpha/SL41. The SL41 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/SL41. Serine protease expressed by pYES2/SL41 was induced by galactose (maximal activity 16.5 units ml(-1) at 40 degrees C, pH 8.0) and was produced in fermentation liquid cultured for 60 h. Northern blot analysis indicated that SL41 transcripts are differentially accumulated at different culture time.

Directing vanillin production from ferulic acid by increased acetyl‐CoA consumption in recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.

Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli.

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.

Isolation of a novel plasmid, pNi15, fromEnterobactersp. Ni15 containing a nickel resistance gene

We isolated nickel-resistant bacterium from soil in order to identify a novel nickel resistance determinant. Using 16S rRNA gene sequencing, an isolate was identified as Enterobacter sp. Ni15. This species showed a medium-level (resistant to up to 10 mM) nickel resistance in nutrient-rich media. Enterobacter sp. Ni15 has a novel plasmid, pNi15, and an increased nickel resistance to Escherichia coli DH5alphain trans. To isolate the nickel resistance gene from pNi15, the plasmid was digested with XbaI and its fragments were cloned into pBluescriptIISK(+). The clones were transferred into E. coli DH5alpha. The nickel resistance of the clones was then assayed. From these results, a pNi15100 isolate containing a 5,328 bp XbaI fragment of pNi15 was identified and sequenced. The E. coli DH5alpha harboring the pNi15100 showed a resistance to up to 7 mM nickel. Using a subcloning analysis, we were able to identify the novel nickel resistance determinant: the nrp gene encoding the putative proteins NrpA and NrpB.

A mutated human tumor necrosis factor-alpha improves the therapeutic index in vitro and in vivo

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has cytotoxic, cytostatic and immunomodulatory effects on malignant tumors. However, clinical trials have revealed high systemic toxicity and this has hampered its utilization as an anti-cancer agent. In this study, a human TNF-alpha mutant was created and tested for its anti-tumor effects. The TNF mutant (recombinant mutated human TNF; rmhTNF) was prepared by protein engineering in which amino acids Pro, Ser and Asp at positions 8, 9 and 10 of TNF-alpha were substituted by Arg, Lys and Arg, and C terminal Leu157 was substituted by Phe, along with deletion of the first seven N-terminal amino acids. Prokaryotic expression recombinant vector pBV-mhTNF containing the PLPR promotor was constructed and transformed into E. coli DH5alpha. The rmhTNF was expressed in a partially soluble form in DH5alpha, purified from the supernatant of cell lysate by ammonia sulfate precipitation and two sequential chromatographic steps. The purified rmhTNF was >95% pure by SDS-PAGE stained with silver and high-pressure size exclusion chromatography (SEC-HPLC). Its yield was about 1.22 mg/g wet cell paste. The mutant rmhTNF exhibited an approximately 50-fold increase in cytotoxicity relative to the wild-type rhTNF on the mouse fibroblast cell line L929 in a standard cytotoxicity test, and at least and at least 50 times higher LD50 as wild type rhTNF in mice. In vivo biological activity studies carried out on tumor cell transplanted mice and nude mice also showed a more effective cytotoxicity of rmhTNF than rhTNF. These results suggest that rmhTNF has potential for developing an effective anti-tumor reagent for some tumors.

Detection of Single Bacterial Pathogens with Semiconductor Quantum Dots

Semiconductor quantum dots (QDs) have been used in a simple fluorometric assay to detect single cells of the pathogenic Escherichia coli O157:H7 serotype. Composed of CdSe/ZnS core/shell QDs conjugated to streptavidin, this system exhibits 2 orders of magnitude more sensitivity than a similar assay using a common organic dye. Selectivity for this pathogenic bacterial strain over a common lab strain (E. coli DH5alpha), which is gained from the use of specific biotinylated antibodies, is also demonstrated for QD labeling. Under continuous excitation, these QDs retain high fluorescence intensities for hours, whereas a typical organic dye bleaches within seconds, allowing for more rapid and accurate identification of E. coli O157:H7 in single-cell fluorescence-based assays. This indirect QD labeling method, based on antibody-antigen and streptavidin-biotin interactions, is flexible enough to expand to other systems and has great potential for use in simultaneous multicolor detection schemes.

Expression of the chitinase gene from Trichoderma aureoviride in Saccharomyces cerevisiae

Chitinase gene ech42 was obtained from Trichoderma aureoviride M and amplified by PCR. The isolated DNA of ech42 was then sequenced. The results showed that the open reading frame of ech42 was 1,447 bp long, encoding 421 amino acids. Three introns were found in the sequence. The cloning vector pMD18-T and an E. coli DH5alpha host were used to yield clones as E. coli DH5alpha/ech42. The ech42 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/ech42. Chitinase expressed by pYES2/ech42 was induced by galactose (maximal activity 0.50 units ml(-1)) and was produced in fermentation liquid cultured for 36 h.

Integration of Metal-Resistant Determinants from the Plasmid of an Acidocella Strain into the Chromosome of Escherichia coli DH5?*

Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5alpha with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523-4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5alpha strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5alpha strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (ZnrAmpr) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmpr E. coli DH5alpha clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5alpha.

2F3 monoclonal antibody recognizes the O26 O-antigen moiety of the lipopolysaccharide of enterohemorrhagic Escherichia coli strain 4276.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) organisms are groups of pathogenic strains whose infections are characterized by a typical lesion of enterocyte attachment and effacement. They are involved in enteric diseases both in humans and in animals, and EHEC strains can be responsible for hemolytic uremic syndrome in humans. Previously, it was shown that the 2F3 monoclonal antibody (MAb) is specific for the O26 EHEC and EPEC strains (P. Kerr, H. Ball, B. China, J. Mainil, D. Finlay, D. Pollock, I. Wilson, and D. Mackie, Clin. Diagn. Lab. Immunol. 6:610-614, 1999). As these groups of bacteria play an important role in pathology, the aim of this paper was to characterize the antigen recognized by the 2F3 MAb and its genetic determinant. A genomic locus containing the entire O-antigen gene cluster and half of the colanic acid gene cluster from an O26 EHEC strain was shown to be sufficient for the production of the antigen recognized by the 2F3 MAb in an E. coli DH5alpha strain. By transposon mutagenesis performed on the recombinant plasmid, all 2F3 enzyme-linked immunosorbent assay-negative mutants had their transposons inserted into the O-antigen gene cluster. The O-antigen gene cluster was also cloned from an O26 EHEC strain into the E. coli DH5alpha strain, which then produced a positive result with the 2F3 MAb. Further analysis of the type of lipopolysaccharides (smooth or rough) produced by the clones and mutants and of the O antigen of the 2F3-positive clones confirmed that the epitope recognized by the 2F3 MAb is located on the O antigen in the O26 EHEC and EPEC strains and that its genetic determinant is located inside the O-antigen gene cluster.

Cloning and expression of pyrroloquinoline quinone (PQQ) genes from a phosphate-solubilizing bacterium Enterobacter intermedium.

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5alpha. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50-95% homology to pqq genes of other bacteria. E. coli DH5alpha expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5alpha expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.

Detection of E. coli DH5alpha specific bacteriophage in the vicinity of a water treatment plant

Air in the vicinity of a water treatment plantscheduled for redevelopment was monitored foraerosols containing a bacteriophagespecific for the Escherichia coli (E.coli) strain DH5alpha bacterium. An assay forthe detection of the specific airbornebacteriophage was developed. Sampling wascarried out prior to and after completion ofdevelopment work to the plant. Preliminaryexperiments showed water from the plantcontains the bacteriophage specific for theE. coli DH5alpha bacterium. Two fieldstudies were completed before work on thetreatment plant began. The first study,conducted under wet and windy conditions, usedonly passive air sampling to monitor air at 3selected locations adjacent to the plant.E. coli DH5alpha specific bacteriophage waspositively detected, albeit at low levels. However, both the passive and a new direct airsampling technique were employed in the secondfield study, conducted under dry and windyconditions. In this instance, neither methoddetected specific bacteriophage. Further workwas undertaken 6 months after modifications tothe plant had been completed. Passive anddirect air sampling techniques were againemployed at each of the same 3 selectedlocations. Samples from both of these methodscontained no bacteriophage. As a check, thesamples were also analysed using an alternativeprocedure. No bacteriophage was detected. Theresults generated indicate that the levels ofairborne specific bacteriophage in the vicinityof the plant appear to be extremely low, andthe direct air sampling technique successful.

Identification of a novel mycobacterial transcriptional regulator and its involvement in growth rate dependence and stringent control.

A novel transcriptional regulator has been identified in the 400-bp upstream region of the guaA gene of Mycobacterium tuberculosis H37Rv that promotes the expression of lacZ gene in Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv but not in Escherichia coli DH5alpha. PCR-mediated deletion mutagenesis and cloning identified a 120-bp fragment upstream from the guaA gene to be the actual regulator. Primer extension analysis mapped the transcription start site to be the first 'G' residue of the translation start codon GTG of the guaA gene. Electrophoretic mobility shift assay showed strong binding of M. smegmatis RNA polymerase holoenzyme to the 400-bp fragment that expresses lacZ in mycobacterial species and a weak binding to the 280-bp fragment that expresses only in E. coli DH5alpha. Both promoter recombinants revealed varied response in the presence of purine nucleotides and exhibited down-regulation when subjected to amino acid starvation.

Urease of enterohemorrhagic Escherichia coli: evidence for regulation by fur and a trans-acting factor.

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5alpha background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Deltafur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.

Identification of genes encoding conjugated bile salt hydrolase and transport in Lactobacillus johnsonii 100-100.

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, alpha and beta, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3' partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5' and 3' deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3' of the cloned segment. The predicted amino acid sequence of the 5' partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5alpha cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [14C]taurocholic acid that were increased as much as threefold over control levels. [14C]Cholic acid was taken up in similar amounts by strain DH5alpha pSportI (control) and DH5alpha p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.

The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows.

A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metalphosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this environmental isolate. Recombinant Escherichia coli DH5 alpha bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO2(2+)) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO2PO4). Subsequently, HUO2PO4-laden cells removed Ni2+ from dilute aqueous flows via intercalation of Ni2+ into the HUO2PO4 lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5 alpha (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5 alpha (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects.

Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli.

Rahnella aquatilis is a Gram-negative bacterium that can fix atmospheric nitrogen and also has the ability to solubilize mineral phosphate. We have cloned the genes that confer the mineral phosphate solubilizing (Mps) trait from this organism by mobilizing a cosmid library of R. aquatilis into Escherichia coli HB101. A 7.0-kb EcoRI fragment from a cosmid, when transferred into E. coli strains HB101 and DH5 alpha, conferred the ability to solubilize hydroxyapatite and the production of gluconic acid to E. coli. The relative amounts of soluble phosphate and gluconic acid produced by the cloned 7.0-kb EcoRI fragment in E. coli were significantly higher than those of R. aquatilis. Nucleotide sequence analysis revealed two complete open reading frames (ORF1 and ORF2) and a partial ORF, ORF1 and ORF2 encoded proteins of molecular mass 10 kDa and 44 kDa. The 44-kDa protein showed extensive sequence similarity to pqqE of Erwinia herbicola, Klebsiella pneumoniae and A. Acinetobacter calcoaceticus. The 10-kDa protein revealed strong similarity to the pqqD of K. pneumoniae and A. calcoaceticus.