Phage Promoter Research Articles

A dual protein expression system in Bacillus subtilis

We have developed a dual expression system for the simultaneous overexpression of two proteins in Bacillus subtilis. Two candidate genes, xylanase ( xynA) and glucanase ( bglS) from B. subtilis strain 168, which were engineered with independent Shine–Dalgarno (SD) sequences, were cloned in tandem into a transfer vector, which was then transformed into B. subtilis strain 1A304 (φ105MU331). The genes were under the transcriptional control of a strong promoter of a bacteriophage, φ105, where transcription was initiated upon thermal induction. Six constructs were made to compare the factors that affected the yields of the gene products. The expression level of each candidate gene was found to correspond to its position relative to the phage promoter, irrespective of the identity of the insert. The lower expression level of the second insert might have been due to limited resources for protein synthesis, a short half-life of the mRNA, or an early termination of the RNA polymerase. Curiously, gene duplications in tandem did not lead to further increase in production.

High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter.

Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia colicells. In system [R], the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter. To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system [R,M]) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system [R+M]). In batch shake flasks containing complex media, co-expression of the methylase gene in systems [R,M] and [R+M] resulted in a 2- and 3-fold increase in volumetric productivity over system [R], yielding activities of 250x10(6) U l(-1) and 350x10(6) U l(-1), which were 28 and 39 times higher than the data in the literature, respectively. Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels. System [R,M] is preferable for high expression levels at longer operation periods, while system [R+M] is well-suited for high expression levels in short-term bioreactor operation.

Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli.

The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.

Reverse transcriptional mutagenesis induced by ribonucleoside triphosphate analogue, PTP and its availability for anti-HIV-1 therapy.

The bicyclic pyrimidine analogue, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one (P) can base pair with both A and G. The riboside 5'-triphsophate of P (PTP) efficiently induces mutation during in vitro transcription and reverse transcription cycles using a phage promoter. In the present study, we have constructed an in vitro transcription system promoted by the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) using HeLa nuclear extract supplemented with HIV-1 Tat protein. Using this system, the effects of mutagenic ribonucleotide analogues were studied.

Bacteriophage-derived genetic tools for use in lactic acid bacteria

Strains of lactic acid bacteria (LAB) are commonly used in industrial fermentations for the production of food products such as cheeses, buttermilk, sauerkraut and yogurt. It has long been recognised that bacteriophages infecting LAB strains can cause serious problems in dairy fermentations, resulting in economic losses and therefore research has been focussed on elucidating the mechanisms by which these bacteriophages proliferate. In recent years, significant advances in molecular biological technologies has allowed researchers to gain an insight into the genetic processes underlying many steps in LAB bacteriophage lifecycles. The knowledge gained from this research has in turn, enabled the development of novel genetic tools utilising phage genes, promoters, and DNA fragments for use in LAB strains, which is the focus of this review.

A general mechanism for viral resistance to suicide gene expression.

Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids. Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort. T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product. Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase. Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved. A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages. A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited intermediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes. Alterations in both RNA polymerase and a second gene are thus responsible for resistance. These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing.

Molecular Cloning and Characterization of the Gene Coding for Azoreductase from Bacillus sp. OY1-2 Isolated from Soil

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Just how does the cII selection system work in Muta™Mouse?

The lambda CII protein is an essential component in the lytic vs. lysogeny decision a bacteriophage makes upon infection of a host at low temperatures. The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection. The Big Blue and Muta Mouse are two widely used in vivo mutational model systems. The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively). The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent. The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue rodent system was first reported by Jakubczak et al. ([1996]: Proc Natl Acad Sci USA 93:9073-9078). More recently, this selection assay has been applied successfully to the Muta Mouse (Swiger et al. [1999]: Environ Mol Mutagen 33:201-207). The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity. The cII selection assay in the Big Blue relies on the presence of the lambda repressor protein CI. In contrast, the recombinant construct used to make the Muta Mouse transgene lacks functional CI protein. Nevertheless, we report an excellent system for quantifying mutations at the cII locus in Muta Mouse. Just how does cII selection work in the Muta Mouse? Written in the context of lambda recombinant genetics, this paper explores the question further.

Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications

BackgroundInfections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage.ResultsHere we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the λimm434 prophage.ConclusionsSimple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains.

RNA Polymerase—Promoter Recognition. Specific Features of Electrostatic Potential of “Early” T4 Phage DNA Promoters

Comparative analysis of electrostatic potential distribution for “early” T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase α-subunit. The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase. A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase α-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.

T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis.

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.

Quantitative Analysis of Aldolase A mRNA in Liver Discriminates between Hepatocellular Carcinoma and Cirrhosis

Chronic liver diseases can progress to cirrhosis and to hepatocellular carcinoma. Timely and unequivocal recognition of the neoplastic evolution of cirrhosis is critical. To this aim, we used a noncompetitive reverse transcription-PCR procedure to analyze aldolase A mRNA in liver tissue from patients with chronic liver diseases at different stages. We studied 12 patients with hepatocellular carcinoma, 19 patients affected by chronic hepatitis C or cirrhosis, and 7 healthy controls. Aldolase A mRNA was reverse-transcribed to cDNA, which was then amplified by PCR. The amplified segments were "read" with a novel dot-blot procedure. A calibrator with the same sequence, synthesized in vitro using a T7 phage promoter, was processed at scalar dilutions in parallel to the target samples to generate a calibration curve and so quantify the target mRNA (detection limit, 0.03 amol; linearity spanning five orders of magnitude). Aldolase A mRNA was approximately 10-fold higher in liver biopsies from patients with hepatocellular carcinoma vs patients with chronic hepatitis C or cirrhosis, and healthy individuals. Furthermore, aldolase A mRNA concentrations were 1.2- to 21.3-fold higher in 12 liver biopsies compared with the paired surrounding cirrhotic tissue. The quantitative analysis of liver tissue aldolase A mRNA differentiates between nonneoplastic chronic liver diseases and hepatocellular carcinoma, which suggests that it has diagnostic potential.

A Method for Demonstrating Gene Essentiality in Staphylococcus aureus

A method for demonstrating whether a gene of Staphylococcus aureus is essential for growth in a rich medium is described. We have used this method to determine whether the murE gene, which encodes the UDP–N–acetylmuramyl tripeptide synthetase required for peptidoglycan synthesis, is essential for growth in S. aureus. In this study, strain CYL368 was constructed from S. aureus RN4220 by placing the murE gene in the chromosome under the control of the spac promoter (a hybrid promoter of the Escherichia coli lac operator and the Bacillus subtilis SPO1 phage promoter). To regulate the murE gene in CYL368, the E. coli lacI gene was expressed from the B. licheniformis penicillinase gene (pcn) promoter in plasmid pMJ8426. Strain CYL368(pMJ8426) grew normally in the presence of isopropyl-β-d-thiogalactopyranoside but could not grow in the absence of the inducer. These results indicate that the murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed for bacterial growth. We concluded that murE is an essential gene of S. aureus.

Changes in the 17 bp spacer in the PR promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation

Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the −10 and −35 consensus regions of the PR promoter of bacteriophage λ. Abortive initiation assays at 37 °C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in kf, the rate constant for conversion from closed (RPc) to open (RPo) complexes, by approximately a factor of 4. The mutation did not significantly affect KB, the equilibrium constant for formation of closed complexes, and decreased KBkf by a factor of 3. Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of kf by a factor of about 25 to 30, and KBkf by a factor of 7 to 8. When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RPo at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of KBkf was about 15 times lower than the corresponding value for two promoters with wild-type spacing.Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 °C between RPi, a stable intermediate in which DNA strands are not separated, and RPo. Although differential sensitivity of single-stranded bases to KMnO4 indicated that in about 20 % of the open complexes at 20 °C the DNA strands are not fully separated (RPo1), the distribution between these complexes and RPo2 (DNA strands fully separated) was also not affected significantly by changes in spacer length. Thus, changes in spacer length primarily affect k2, the rate constant for conversion of RPc to RPi, which corresponds to a nucleation of DNA strand-separation.Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between PR derivatives with 16 bp spacing and those with 18 bp spacing.

The Legionella (Fluoribacter) gormanii metallo-beta-lactamase: a new member of the highly divergent lineage of molecular-subclass B3 beta-lactamases.

A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the bla(FEZ-1) gene in E. coli, based on the T7 phage promoter, was also developed.

Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system.

The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.

Macrophomate Synthase: Characterization, Sequence, and Expression in Escherichia coli of the Novel Enzyme Catalyzing Unusual Multistep Transformation of 2-Pyrones to Benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.

Analysis of the Phenotypic Effect of the ts47 Mutation of the Pseudomonas aeruginosa Transposable Phage D3112 on the Expression of Late Phage Genes

At nonpermissive temperature (42°C) thets47 mutation causes substantial abnormalities in the late phase of the phage intracellular development. In these conditions DNA of the D3112 phage is detected both in a free form and integrated into bacterial chromosome. The transcription kinetics in the ts47 mutant at 42°C was indistinguishable from that typical to other early gene mutants (A, B, and C): specifically, the preservation of the first transcription peak along with low activity of late transcription were observed. Similarly to the C gene, the ts47 mutation-carrying locus is involved in regulating the transcription of the D3112 transposable phage late genes. It is suggested that the mechanism underlying the action of thets47 mutation differs from that of the C gene product. One of the possible explanations is based on the fact that the product of the ts47 locus affects the activity of cellular RNA polymerase via providing more effective recognition of the phage promoters by the RNA polymerase modified with the phage protein C.

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker.

We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase--a selectable nuclear marker--in Chlamydomonas reinhardtii. The 5' end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8phi3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed.

Genetic engineering of Bacillus subtilis for the commercial production of riboflavin

Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5′ end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway.