Translational Fusion Vector Research Articles

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families.

The expression pattern of the genome in Escherichia coli is controlled by regulating the utilization of a limited number of RNA polymerases between a total of 4600 genes on its genome. The distribution pattern of RNA polymerase on the genome changes after two steps of protein-protein interaction with seven sigma subunits and about 300 transcription factors (TFs). Based on a systematic search for the regulation target promoters recognized by each TF, we propose two novel concepts: each TF regulates a number of target promoters; and each promoter is regulated by many TFs. In parallel, attempts have been made to determine the intracellular concentrations of all TFs using two systems: quantitative immunoblot analysis using TF-specific antibodies; and reporter assay of TF promoter activities. The direct measurement of TF protein level has so far been published for a set of 60 regulators with known functions. This study describes the determination of growth phase-dependent expression levels of 90 TFs using the reporter assay system. The translational fusion vector was constructed from the TF promoter sequence including an N-terminal proximal TF segment and the reporter GFP. At the beginning of cell growth, high-level expression was observed only for a small number of TFs. In the exponential phase, approximately 80 % TFs are expressed, but the expressed TF species change upon transfer to the stationary phase. Significant changes in the pattern of TF expression were observed between aerobic and anaerobic conditions. The list of intracellular levels of TFs provides further understanding to the transcription regulation of the E. coli genome under various stressful conditions.

A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs

Bacteriophage vectors for achieving single-copy gene expression linked to a colorigenic reporter assay have been used successfully for genetic screening applications. However, the limited number of cloning sites in these vectors, combined with the requirement for lac- strains and the time- and/or media-dependence of the chemical-based colorimetric reaction, have limited the range of applications for these vectors. An alternative approach using a fluorescent reporter gene such as green fluorescent protein (GFP) or GFP derivatives could overcome some of these technical issues and facilitate real-time monitoring of promoter and/or protein activity. Here, we report the development of a novel translational bacteriophage fusion vector encoding enhanced GFP (eGFP) that can be incorporated into the chromosome as a single-copy gene. We identified a Bacillus promoter (BP) that is stably expressed in Escherichia coli and drives ~6-fold more expression of eGFP than the T7 promoter in the absence of inducer. Incorporating this BP and RNase III target signals into a single system enabled clear detection of the absence or downregulation of RNase III activity in vivo, thereby establishing a system for screening and identifying novel RNase III targets in a matter of days. An RNase III target signal identified in this manner was confirmed by post-transcriptional analysis. We anticipate that this novel translational fusion vector will be used extensively to study activity of both interesting RNases and related complex or to identify or validate targets of RNases that are otherwise difficult to study due to their sensitivity to environmental stresses and/or autoregulatory processes.

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter.

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin.

The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.

A refined vector system for the in vitro construction of single-copy transcriptional or translational fusions to lacZ

New single-copy vectors based on λ phage have been developed for creating either transcriptional (operon) or translational (gene) fusions to the lacZ gene. The improvements of these vectors over the previous λTL61 vector include: (i) incorporation of a tetracycline-resistance-encoding gene (Tc R) to permit direct selection of lysogens, ( ii) low-background β-galactosidase activity, ( iii) the ability to accept DNA inserts up to 8 kb in size, and ( iv) an expanded multiple cloning site (MCS). The new transcriptional fusion vector retains the RNase III processing site downstream from the MCS which ensures independent translation of lacZ. The set of three translational fusion vectors allow for convenient subcloning in any of the three translational reading frames

Activity of mycobacterial promoters during intracellular and extracellular growth.

pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.

Relative strengths of promoters from Cochliobolus heterostrophus

GPD1 (encoding glyceraldehyde-3-phosphate dehydrogenase) is a constitutively expressed gene in Cochliobolus heterostrophus that produces a single transcript. The steady state level of GPD1 mRNA is 14-fold greater than that of the constitutively-expressed TRP1 gene (encoding a tryptophan biosynthesis enzyme) indicating that GPD1 has a stronger promoter and/or a more stable mRNA. A set of lacZ translational fusion vectors was constructed to compare the gene expression signals of GPD1, TRP1 and PRO1 (a C. heterostrophus genomic fragment selected for promoter activity) in C. heterostrophus as single copies at the same site in the chromosome. Under conditions that repressed endogenous beta-galactosidase expression, beta-galactosidase activity in transformants was constitutive and required the GPD1, TRP1 or PRO1 expression signals. In-frame GPD1::lacZ activities were 6-fold greater than in-frame TRP1::lacZ and PRO1::lacZ activities, indicating that GPD1 has more efficient expression signals.

An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus.

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed. This assay indicated that delta CenA activity was largely independent of cellular location. To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70%. One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C. crescentus than linkage of the RsaA N terminus. Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, delta CenA possessed many of the attributes of an "all-purpose" reporter.

In vivo translational start site selection on leaderless mRNA transcribed from the Streptomyces fradiae aph gene.

The message of the Streptomyces fradiae aph gene lacks a leader sequence and therefore is translated in the absence of a conventional Shine-Dalgarno interaction between mRNA and 30S ribosomal subunits. Insertion mutations generating short leaders of 2 or 4 nucleotides on the 5' end of the aph transcript reduced translational efficiency. A 4-base leader (5'-AUGC-3') placing a potential out-of-frame start codon immediately upstream of the aph coding sequence prevented detectable translation in the aph reading frame. The upstream AUG in this mutant was able to drive the expression of a reporter gene in a translational fusion vector, implying that this start codon was utilized in favor of the downstream AUG. Additional leaders (5'-AUAUGC-3' or 5'-CAUAUGC-3') placing 2 or 3 nucleotides 5' to the upstream AUG relieved this apparent discrimination, permitting translation of the APH protein from the downstream AUG. These results suggest that the position of a start codon with respect to the 5' terminus of aph mRNA is a determinant of translational efficiency and start site selection.

In vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana.

Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.

Use of alacZfusion to study transcriptional regulation of theRhodobacter capsulatus hemAgene

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for Gram-negative bacteria

A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters ( p Nm, plac, ptac and p S1 ) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by β-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the Nm R promoter. The Gm R gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, p Gm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.

Analysis of the fowlpoxvirus gene encoding the 4b core polypeptide and demonstration that it possesses efficient promoter sequences

The gene encoding the fowlpox virus 4b core polypeptide has been identified by analogy with the vaccinia 4b gene. It has been cloned, and its nucleotide sequence determined. The gene, which is 1971 nucleotides long, can encode a protein of 75,200 Da (75.2K polypeptide), slightly longer than its vaccinia counterpart with which it shares 52% identity. Sequences upstream of the fowlpox virus 4b gene correspond to the consensus sequence determined for vaccinia late promoters, suggesting that late promoter signals may be shared by the different genera of poxviruses. Upstream sequences have been cloned into a β-galactosidase translational fusion vector and shown to promote the efficient expression of β-galactosidase in a transient assay system. This expression was abolished in the presence of araC, an inhibitor of DNA replication which blocks late gene expression in poxviruses. The fowlpoxvirus 4b promoter should be a useful component of genetically engineered fowlpox virus vaccines.

Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans".

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.

Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumonia nifA protein but not by the product of ntrC.

Rhizobium japonicum nifH'- and nifD'-'lacZ fusions were constructed using the translational fusion vector pMC1403. beta-Galactosidase activities from these fusion plasmids were measured in wild-type, ntrA- and delta(ntrBC) Escherichia coli strains carrying plasmids which overproduced the Klebsiella pneumoniae nifA or ntrC gene products. In contrast to results reported in R. meliloti (ref. in the text) neither nifH nor nifD promoters were activated by the ntrC product. In the presence of nifA gene product, however, beta-galactosidase activity from both nifH and nifD fusion plasmids increased substantially. NifA-mediated activation of these Rhizobium promoters was temperature sensitive and was dependent on the host ntrA product. In order to determine the point at which the fusion transcripts were initiated, RNA was extracted from the wild-type E. coli strain carrying each of the R. japonicum fusion plasmids plus the nifA overproducing plasmid. This RNA was used to perform S1 mapping experiments. NifA-mediated transcription from both R. japonicum promoters, began at the same point as previously determined in soybean root-nodule bacteroids (ref. in the text). The results obtained suggest that there may be differences in the mode of regulation between members of the fast- and slow-growing rhizobia. Also, the results of the S1 mapping experiments indicate that activation of the R. japonicum nitrogenase structural genes may be similar to the activation of nif genes in K. pneumoniae thus raising the possibility that R. japonicum may contain nifA and ntrA-like genes.