Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Human immunodeficiency virus type 1 Tat induces B7-H1 expression via ERK/MAPK signaling pathway

In HIV-infected subjects, B7-H1 synthesis and expression are up-regulated, and the degree of dysregulation correlates with the severity of disease. HIV-1 Tat protein, the viral transactivating factor, represents a key target for the host immune response. However, the relationship between B7-H1 and Tat protein has not been addressed. Here, we chose human endothelial cells which provide costimulatory signals sufficiently to influence T cells. We used recombinant pcDNA3.1(+)–Tat plasmid to transfect human endothelial cells ECV304 to establish stable Tat-expressed cell strain, and found that HIV-1 Tat was able to induce B7-H1 expression in ECV304 cells by Real-time PCR and flow cytometry analysis, and inhibited lymphocyte proliferation in co-culture system. Moreover, by using pharmacological inhibitor of ERK pathway, HIV-1 Tat induces B7-H1 expression via ERK/MAPK signaling pathway was corroborated. In summary, our results indicate that HIV-1 Tat could induce B7-H1 synthesis in ECV304 cells through ERK/MAPK signaling pathway.     

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.     

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.     

Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.     

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the PR promoter of bacteriophage lambda

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.     

Identification of a protein coded by pR plasmid and involved in SOS repair in E. coli.

The TP120 plasmid is known to determine enhanced UV survival in E. coli wild type an uvrB and PolA mutants but not in RecA mutant. In order to analyze the function involved in the SOS repair, we have constructed a new plasmid named pR derived by cleavage of TP120 with Hind III endonuclease. This new plasmid maintains the Ap and UV resistance. The insertion of Tn5 transposon in the plasmid allows to select several pR::Tn5 plasmids whose UV resistance was inactivated by the transposition. The comparison of the protein synthesis in the minicells of the pR and pR::Tn5 shows that the pR codes for a 22.000 M.W. dalton protein which is absent in protein pattern of pR::Tn5.     

A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803

A vector-host system for testing promoters in the cyanobacterium Synechocystis PCC6803 has been constructed. It relies on a small Escherichia coli promoter-probe plasmid, pFF11, which has four unique restriction sites in a polylinker upstream from the cat reporter gene. This plasmid is able to obtain a cyanobacterial origin of replication by homologous recombination with the resident plasmid of the recipient host, generating a new E. coli-Synechocystis PCC6803 shuttle vector. This plasmid does not confer any detectable chloramphenicol acetyl transferase activity to this cyanobacterium in the absence of a promoter insert. Several heterologous promoters were tested in Synechocystis PCC6803 using this system. Results obtained with the lambda pR promoter and the repressor-encoding cI857 gene demonstrate that these elements can be used for high-level and tightly regulated gene expression in Synechocystis PCC6803.     

The rep region of pR plasmid regulates the expression of SOS system

Summary By using an artificial hybrid between phage and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes was monitored by a fusion between the muc promoter and the lacZ structural gene. When E. coli cells containing such a fusion are infected by the hybrid pR phasmid, -galactosidase activity is enhanced, indicating that pR encodes an antagonist of lexA. By deletion mapping we have located the gene encoding the antagonist of lexA (bat) in the rep region of the plasmid. The bat gene product can also antagonize the cI repressor as shown by the observation that pR phasmids are virulent on a homoimmune lysogen. We have exploited this latter property to carry out genetic and functional analysis of the bat region. This region is organized as a classical operon where the expression of the bat structural gene is negatively regulated by a repressor gene that encodes a proteic product.     

Alteration of phagocyte functional activity depending on the presence of pR50 plasmid in bacteria

The relationship between the presence, or the absence, of conjugative plasmid pR50 detected in Klebsiella oxytoca 89, in the isogenic pairs of attenuated strains Shigella flexneri 2a 516 Near and Salmonella typhimurium 129 Rifr and the regeneration of the active forms of oxygen by mouse peritoneal phagocytizing cells was studied. As indicated by the data obtained in the course of the experiment, plasmids pR50 could influence the chemiluminescent response of phagocytes. The inhibition of the synthesis of oxygen metabolites was found to occur at the primary stage of the infectious process, that later this synthesis increased, that facilitated the survival of the animals infected with the cultures carrying plasmid pR50.     

Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli

Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.