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.     

Construction and characterization of a cosmid of Streptomyces lividans

Summary We constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).     

Influence of plasmid pR50 controlling bacterial protease activity on Salmonella experimental infection

The comparative analysis of the infectious process and immunological parameters in (CBA x C57BL/6)F1 mice infected with S. typhimurium isogenic strains differing by the presence of plasmid pR50 determining protease activity, was carried out. A growth in the expression level of antilactoferrin, anticomplementary and anti-immunoglobulin activity in bacteria isolated from the spleen in the course of the infectious process was detected. In mice infected with S. typhimurium having plasmid pR50, in contrast to nonplasmid recipients, a higher level of contamination of organs, the suppression of spontaneous, stimulated production of interferon-gamma and the bactericidal properties of peritoneal macrophages were noted. The data obtained in this investigation suggested that the acquisition of R-plasmid of 50 MD, controlling protease activity and multiple medicinal resistance, contributed to the persistence of intracellular bacteria.     

Expression in Escherichia coli of an immunoreactive visna virus transactivating protein

We have cloned the gene encoding the visna 1514 transactivating protein, Tat, into the Escherichia coli lambda pR expression plasmid, pRIT2T. Efficient synthesis of the protein A::Tat fusion protein was obtained in host strains which carried either wild-type or temperature-sensitive (ts) lambda repressors. However, constitutive synthesis of the fusion protein in these host strains resulted in selection against plasmids which synthesized the fusion protein. Efficient repression of the lambda pR promoter was obtained using a ts repressor gene carried on a multicopy plasmid. Synthesis of the fusion protein in this strain was efficient on induction, and reproducible after subculture. Antisera generated against the termini of visna Tat were used to demonstrate that the fusion protein retained the antigenicity of both the N and C termini of the transactivating protein.     

The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival.

It has been recently reported that the pR plasmid enhances the UV survival in E.coli c600. In order to test whether this function may be expressed also in mammalian cells, LTA (tk- aprt-) mouse cells were cotransformed with pR plasmid DNA and ptk1 plasmid as selectable marker. Tk+ transformants were analyzed for their UV survival and for the presence of pR DNA sequences by blot-hybridization. The results show a correlation between the enhanced UV survival and presence of pR DNA sequences in cotransformed LTA mouse cells.     

Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli.

The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells. Statistical analysis showed that the expression of the uvp1 and uvp2 regions significantly increased (P less than 0.01) the survival upon exposure to UV light in mouse cells and bacteria. These results might suggest the presence of an inducible repair response to DNA damage in mouse LTA cells.     

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk- aprt-) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline-N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl-N'-nitro-N-nitrosoguanidine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents. The possible interactions between pR plasmid products and the different repair enzymes involved are discussed.     

Evolution and utility of a Pseudomonas aeruginosa drug resistance factor.

We describe the addition to the Pseudomonas aeruginosa sex factor, FP2, of carbenicillin resistance encoded by the RP1 plasmid. This occurred in a step-wise manner as detected by variations in the characteristics of the FP2-RP1 plasmid aggregate. The addition of the carbenicillin resistance marker to FP2 facilitates estimates of FP2 transfer. Transfer frequencies for the presumed cointegrate plasmid, using carbenicillin selection, approached 10(-1) per donor bacterium. The chromosomal mobilization properties of the derived plasmid, designated pR0271, resembled those of the progenitor plasmid FP2. Plasmid pR0271 was also observed to mobilize a nontransmissible drug resistance plasmid sharing genetic homology at frequencies corresponding to those observed for chromosomal markers proximal to the origin of transfer.     

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.     

Design of Streptomyces nogalater LV65 strains with higher synthesis of nogalamicin using regulatory genes

Influence of cloned regulatory genes on biosynthesis of nogalamicin by Streptomyces nogalater LV65 strains has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into the cells of wild type strain of S. nogalater LV65 resulted in higher synthesis of nogalamicin. A similar effect was observed at heterologous expression of gene ppGpp of synthetase relA cloned in S. coelicolor A3(2). Heterologous expression of genes absA2from S. ghanaensis ATCC14672 and lndyR from genome S. globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.     

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)     

A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid

Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.     

A plasmid vector with temperature-controlled gene expression

A 169 b.p. fragment including the bla gene promoter p3 has been removed from pBR327 plasmid, and the deleted plasmid used for cloning the TaqI/BglII-fragment of the lambda c1857ind- DNA containing promoter pR and gene cI to obtain plasmid pCE119. Cells containing pCE119 produced a high level of beta-lactamase at 42 degrees C, the yield at 42 degrees C being 100 times higher than at 32 degrees C. For cloning and functional assays a pCEZ12 plasmid was constructed, in which promoter pR and repressor cI of lambda phage control the expression of the semi-synthetic beta-galactosidase gene. Yield of beta-galactosidase produced by pCEZ12 at 42 degrees C was ca. 300 times higher than at 32 degrees C.     

Salmonella enterica Serovar Typhi Plasmid Impairs Dendritic Cell Responses to Infection

Salmonella enterica serovar Typhi (S. typhi) evades from innate immunity by expression of a variety of pathogenic factors. The "pR(ST98)" plasmid of S. typhi is involved in multidrug-resistant and virulence of S. typhi. However, its exact effect on host cell function remains elusive. Dendritic cells (DCs) play an important role in shaping immune response against Salmonella. For the purpose of investigation whether pR(ST98) might target DCs involved in adaptive immune response, murine DCs were infected with S. typhi wild type and mutant strains. S. typhi stimulation resulted in up-regulation of costimulatory molecules on DCs. S. typhi wild type resulted in decreased up-regulation of CD40, CD80, and CD86 expression. Experiments with S. typhi pR(ST98) mutant (S. typhi-Δ-pR(ST98)) and S. typhi-Δ-pR(ST98) with a complemented plasmid encoding pR(ST98) (S. typhi-c-pR(ST98)) revealed that pR(ST98) accounts for inhibition of surface molecule expression and functional maturity. S. typhi-Δ-pR(ST98) gave maximal levels of IL-12 and IFN-γ release compared with wild type S. typhi or the complemented strains. In contrast to IL-12 and IFN-γ, IL-10 secretion by S. typhi-Δ-pR(ST98)-infected DCs was significantly lower than induction by S. typhi wild type. This indicates that immunity in response to pR(ST98) is skewed away from a protective Th1 response. Moreover, infection with S. typhi-Δ-pR(ST98) induced autophagy in DCs. We herein demonstrate S. typhi pR(ST98) plays essential roles in modulating DCs maturation, activation, inflammatory responses, and autophagy. Together, these data prove that pR(ST98) targets functions of DCs that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by S. typhi.