Review: Biosafety of E. coli β-glucuronidase (GUS) in plants

The -glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers     

DNA Packaging by λ-Like Bacteriophages: Mutations Broadening the Packaging Specificity of Terminase,the λ-Packaging Enzyme

The DNA-packaging specificities of phages λ and 21 depend on the specific DNA interactions of the small terminase subunits, which have support helix-turn-recognition helix-wing DNA-binding motifs. λ-Terminase with the recognition helix of 21 preferentially packages 21 DNA. This chimeric terminase''s ability to package λDNA is reduced ∼20-fold. Phage λ with the chimeric terminase is unable to form plaques, but pseudorevertants are readily obtained. Some pseudorevertants have trans-acting suppressors that change codons of the recognition helix. Some of these codons appear to remove an unfavorable base-pair contact; others appear to create a novel nonspecific DNA contact. Helper-packaging experiments show that these mutant terminases have lost the ability to discriminate between λ and 21 during DNA packaging. Two cis-acting suppressors affect cosB, the small subunit''s DNA-binding site. Each changes a cosB^λ-specific base pair to a cosB²¹-specific base pair. These cosB suppressors cause enhanced DNA packaging by 21-specific terminase and reduce packaging by λ-terminase. Both the cognate support helix and turn are required for strong packaging discrimination. The wing does not contribute to cosB specificity. Evolution of packaging specificity is discussed, including a model in which λ- and 21-packaging specificities diverged from a common ancestor phage with broad packaging specificity.VIRUSES must package viral chromosomes from nucleic acid pools that include host-cell nucleic acids, so specific recognition of the viral nucleic acid is essential during virion assembly. For large DNA viruses, including the tailed double-strand DNA (dsDNA) bacteriophages, the herpesviruses, and the adenoviruses, DNA-packaging proteins recognize specific sequences on the viral chromosomes (reviewed in Baines and Weller 2005 and Ostapchuk and Hearing 2005, respectively). For the dsDNA viruses that produce virion chromosomes by processing concatemeric DNA, a viral terminase enzyme functions in the recognition and cutting of concatemeric DNA and subsequently sponsors DNA translocation. λ-Terminase is a heterooligomer of large and small subunits, gpA and gpNu1, respectively. Cutting of concatemeric DNA is carried out by gpA''s endonuclease activity (Becker and Gold 1978; Davidson and Gold 1992; Hwang and Feiss 1996). Three DNA subsites, cosQ, cosN, and cosB, are contained in the ∼200-bp-long cos site and orchestrate DNA packaging through interactions with terminase (Figure 1A; reviewed in Feiss and Catalano 2005). gpA introduces staggered nicks in cosN to generate the 12-bp cohesive ends of mature λDNA molecules. Efficient and accurate nicking of cosN requires anchoring of gpA by gpNu1, which binds to the adjacent cosB subsite (Higgins and Becker 1994b; Hang et al. 2001).Open in a separate windowFigure 1.—The cos and terminase region of the λ-chromosome. (A) (Top) Map of cos and the terminase-encoding Nu1 and A genes. The black bar indicates the location of the winged helix-turn-helix DNA-binding motifs in the N-terminal domain of gpNu1. (Bottom) cos subsites: cosQ is required for termination of DNA packaging; cosN is the site where the large terminase subunit, gpA, introduces staggered nicks to generate the cohesive ends of virion DNA molecules; and cosB contains the gpNu1-binding sites R1, R2, and R3 along with the IHF-binding site I1. (B) (Top) Schematic of gpNu1 residues 1–42, including the support (blue) and recognition (red) α-helixes and the wing loop (magenta). β1 and β2 are short β-strands flanking the DNA-binding elements. (Bottom) Sequences are a comparison of residues of λ''s gpNu1 and phage 21''s gp1, with conserved resides indicated by vertical lines. Note that the recognition helixes of gpNu1 and gp1 differ by four residues, all likely solvent-exposed (Becker and Murialdo 1990; de Beer et al. 2002). (C) Three-dimensional structure of the winged helix-turn-helix-containing, N-terminal domain of gpNu1 (residues 1–68) (de Beer et al. 2002). Side groups of solvent-exposed residues of the recognition helix are displayed. Color coded as in B.λ''s cosB (cosB^λ) is a complex subsite containing three copies of a gpNu1-binding sequence, the R sequence, plus a site, I1, for the integration host factor (IHF), the Escherichia coli DNA-bending protein. The order of sites is cosN–R3–I1–R2–R1. The amino-terminal half of gpNu1 contains a winged helix-turn-helix DNA-binding motif (Figure 1, B and C; Gajiwala and Burley 2000) that interacts with the R sequences. Further, the amino-terminal domain of gpNu1 is a tight dimer (Figure 1C, de Beer et al. 2002). The IHF-induced bend at I1 creates a DNA hairpin in cosB that positions the major grooves of R3 and R2 to face inward, so that the helix-turn-helix motifs of dimeric gpNu1 can be docked into them. The wing loops are positioned to make minor groove contacts with R3 and R2. Thus it is proposed that gpA is positioned to nick cosN by assembly of a bent structure with dimeric gpNu1 bound to R3 and R2 (Becker and Murialdo 1990; de Beer et al. 2002). A variety of studies indicate that the positioning of gpNu1 at R3 is crucial and that the other interactions function to create and/or stabilize the R3–gpNu1 interaction (Cue and Feiss 1993a; Higgins and Becker 1994a; Hang et al. 2001).DNA packaging initiates when terminase binds and nicks a cos. Following cosN nicking and separation of the cohesive ends, terminase remains bound to the cosB-containing chromosome end (Becker et al. 1977; Yang et al. 1997). The DNA-bound terminase docks on the portal vertex of a prohead, the empty, immature virion head shell. Assembly of the ternary prohead–terminase–DNA complex activates gpA''s potent translocation ATPase, and the viral DNA is translocated into the prohead (Yang and Catalano 2003; Dhar and Feiss 2005). Translocation brings the next cos along the concatemer to the portal-docked terminase (Feiss and Widner 1982). The downstream cos is cleaved by terminase, completing packaging of the chromosome. Recognition of the downstream cos requires cosQ and cosN (Cue and Feiss 2001). Following DNA packaging, terminase undocks from the filled head. Attachment of a tail to the DNA-filled head completes virion assembly. The undocked terminase remains bound to and sponsors the packaging of the next chromosome along the concatemer.The interactions between the recognition helix of gpNu1 and an R sequence are typical for helix-turn-helix proteins, as shown by genetic studies of chimeras between λ and its relative, phage 21, as follows: λ and 21 have similarly organized cos sites; the cosB of 21 also has the R3–I1–R2–R1 structure. Nevertheless, the two phages have distinct packaging specificities. Base-pair differences in the R sequences account for packaging specificity (Becker and Murialdo 1990; Smith and Feiss 1993). cosN and cosQ are interchangeable between λ and 21 (Feiss et al. 1981). The consensus R sequences are 5′-CGTTTCCtTTCT-3′ for cosB^λ and 5′-CaTGTCGGncCT-3′ for cosB²¹, where capitalized residues are conserved in all three R sequences of both phages; underlined and capitalized are two residues conserved in all three R sequences of both phages, but which differ between cosB^λ and cosB²¹ (Becker and Murialdo 1990). These two conserved but phage-specific base pairs are likely to be of major importance for specificity. Similarly, the recognition helixes of the helix-turn-helix motifs of the small subunits of λ (gpNu1) and 21 (gp1) terminases differ in four amino acid residues that account for packaging specificity (Figure 1; Becker and Murialdo 1990).In earlier work (de Beer et al. 2002), we showed that modifying λ-terminase by replacing the gpNu1 recognition helix with that of 21''s gp1 created a terminase (gpNu1^hy1 terminase) that was specific for the cosB of phage 21 (designated cosB²¹). That is, λ cosB²¹ Nu1^hy1 was viable, but λ cosB^λ Nu1^hy1 was inviable due to the specificity mismatch between cosB^λ and the cosB²¹-specific recognition helix of the chimeric small terminase subunit, gpNu1^hy1. The Nu1^hy1 terminase packages cosB²¹ chromosomes ∼10-fold more efficiently than it does cosB^λ chromosomes. This 10-fold discrimination between cosB²¹ and cosB^λ chromosomes is much weaker than the >10⁴-fold discrimination shown by wild-type λ and 21 terminases (de Beer et al. 2002). Because of the modest discrimination of Nu1^hy1 terminase, the yield of λ cosB^λ Nu1^hy1 is only slightly below the yield required for plaque formation. Lysates of λ cosB^λ Nu1^hy1 contain plaque-forming pseudorevertants at a level expected for single mutations. A number of these pseudorevertants were sequenced and found to contain mutations in cosB^λ or in the Nu1^hy1 gene. Here we report on in vivo packaging studies on the effects of these Nu1^hy1 and cosB^λ suppressor mutations on packaging specificity.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

Genetic Functions Promoting Homologous Recombination in Escherichia coli: A Study of Inversions in Phage λ

We have studied homologous recombination in a derivative of phage lambda containing two 1.4-kb repeats in inverted orientation. Inversion of the intervening 2.5-kb segment occurred efficiently by the Escherichia coli RecBC pathway but markedly less efficiently by the lambda Red pathway or the E. coli RecE or RecF pathways. Inversion by the RecBCD pathway was stimulated by Chi sites located to the right of the invertible segment; this stimulation decreased exponentially by a factor of about 2 for each 2.2 kb between the invertible segment and the Chi site. In addition to RecA protein and RecBCD enzyme, inversion by the RecBC pathway required single-stranded DNA binding protein, DNA gyrase, DNA polymerase I and DNA ligase. Inversion appeared to occur either intra- or intermolecularly. These results are discussed in the framework of a current molecular model for the RecBC pathway of homologous recombination.     

E. coli DNA replication in the absence of free β clamps

During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps.     

Native supercoiling of DNA: The effects of DNA gyrase and ω protein in E. coli

Molecular Genetics and Genomics - This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrB ts) and a deletion of the top gene...     

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8

Expression of the human Ke 6 gene, 17β-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5α-dihydrotestosterone, and 5-androstene-3β,17β-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4–5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.     

Pressure-induced inactivation of E. coli β-galactosidase: influence of pH and temperatur

In order to assess the feasibility of a high-pressure immunodesorption process using a β-galactosidase-anti-/3-galactosidase complex as a model, the influence of high hydrostatic pressure on the inactivation of E. coli /3-galactosidase has been investigated. The irreversible activity loss of β-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of the phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of β-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using /3-galactosidase as a tag, are discussed.     

A novel assay for illegitimate recombination in Escherichia coli: Stimulation of λbio transducing phage formation by ultra-violet light and its independence from RecA function

We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of λ Spi⁻ phages formed during prophage induction was measured with an E. coli P2 lysogen as the indicator bacteria. Since almost all of the λ Spi⁻ phages thus detected contain attR, they have essentially the same structures as λbio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end. The following results on the formation of specialized transducing phages have been obtained by this assay system to date. (1) Irradiation with UV light greatly enhanced the formation of λ Spi⁻ phages. (2) Treatments with other DNA-damaging agents also enhanced the formation of λ Spi⁻ phages. (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of λ Spi⁻ phage formation is not controlled by the SOS regulatory system. (4) Preliminary results suggested that DNA gyrase is involved in the formation of λ Spi⁻ phage during prophage induction. Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi⁻ assay system can provide important clues to the mechanism of illegitimate recombination.