Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation

The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.     

Entry exclusion determinant(s) of IncN plasmid pKM101.

pKM101 renders its host a poor recipient in conjugal matings with genetically distinguishable derivatives of itself. The gene(s) primarily responsible for this, denoted eex, is located in between genes required for both conjugal transfer and sensitivity to donor-specific bacteriophage, although it itself is not necessary for transfer. A gene linked to, or coincident with, the region needed for vegetative plasmid replication also inhibited establishment of related plasmids under certain conditions. Construction of an operon fusion between eex and the Escherichia coli lac promoter has shown that this gene is transcribed in a clockwise fashion on the circular map of pKM101. To date, we have not been able to visualize a protein product(s) of the eex gene(s).     

The muc genes of pKM101 are induced by DNA damage

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.     

Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.     

Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens

The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens TI plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.     

Functional organization of plasmid pKM101.

Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Analysis of two genes encoding heat-labile toxins and located on a single Ent plasmid from Escherichia coli.

A clinical isolate of Escherichia coli harbored two copies of the heat-labile toxin (LT)-encoding gene (elt) on a 157-kb plasmid. The arrangement of the gene copies is different from the cholera toxin-encoding gene duplication described for some strains of Vibrio cholerae. The nucleotide sequences of the elt alleles are not identical (differing by 2 bp) and the duplicated region flanking the alleles extends 314 bp on one side and 1122 bp on the other side of each copy. Different partial copies of IS600 were identified 280 bp 3' to the stop codon of each allele. Partial and complete copies of other IS were also identified near the elt alleles. The data suggest that the regions surrounding the genes are hot spots for IS which would account for the observed heterogeneity in DNA flanking elt genes.     

The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation)

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.     

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). We have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA⁺ strain but more than pKM101 in a uvrA⁻ strain. muc⁻ point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. We have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB.     

An alteration leading to loss of ability to support phleomycin mutagenesis in the pKM101-derived plasmid pGW16 is located in or close to the mucAB genes

The differences between the plasmid pKM101 and its derivative pGW16, which has lost the ability to support muc-dependent phleomycin mutagenesis, while retaining other muc-dependent phenotypes, have been further investigated. Deletion derivatives which retain only 10.8 kb (approximately one third) of the pKM101 genome but retain the muc genes have been constructed from both pKM101 and pGW16. The deletion derivatives confer protection and mutagenesis-enhancing properties similar to those of their respective parents, indicating that the lesion in the mutant plasmid pGW16 lies in or close to the muc genes. Differences in the muc-dependent phenotypes of strains containing pKM101 or pGW16 suggest that the pGW16 lesion results in either differential loss of function in the muc gene products, or constitutive expression of the muc gene products.     

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.     

Effect of pKM101 plasmid on lethal and mutagenic damage in UV-irradiated E. coli strains

Introduction of the R-factor plasmid pKM101 increased resistance to UV-killing in uvr lexA(Ind-) recA+ strains of E. coli K12 as well as B, while their UV mutability was not affected. Similar effects were also observed in those strains when the 18-B plasmid (a pBR322 derivative carrying the region (about 5 kb) of the 35.4 kb pKM101 plasmid) was introduced. The muc genes which are considered to be involved in error-prone repair are contained in 18-B. These results suggest the possibility that the pKM101 effect requires the host recA gene and a common genetic region, including the muc genes, in both plasmids and is associated with some unmutable repair systems.     

Induction of presumed somatic gene mutations in mice by 2-naphthylamine

Plasmid pKM101, whose mucA and B genes endow cells with enhanced mutation frequency and enhanced resistance to far-ultraviolet radiation (FUV) (254 nm), had no influence on these properties when cells were damaged by near-ultraviolet radiation (NUV) (300-400 nm). Thus, NUV lesions did not lead to induction of SOS repair and subsequent expression of mucA and B genes on plasmid pKM101. Further, when cells were pre-irradiated with NUV and subsequently irradiated with FUV, there was a blockage of SOS repair, including the repair normally controlled by genes on pKM101.     

肠毒素型腹泻活疫苗菌株的构建

将不耐热的肠毒素基因克隆,在限制性内切酶Xba I位点加入4对核苷酸,由于密码阅读框移位,使肠毒素的活力中心A亚单位结构改变,而具抗原性的B亚单位仍保持原状。改造后的肠毒素基因与载体pBR322、pGA22的一部分及pED100的tra基因相连接,得质粒pBRC21、pPMC4及pPMC5,均可指导合成无毒肠毒素LTA~-B~+,其抗原效价为天热肠毒素菌株的30倍左右。用转化或诱动的方法将pPMC4引入从猪分离并能在猪肠道内滞留定居的菌株中;大部分菌株用接合的方法引入pPMC5,共得30多个株菌,可用于制备猪痢口服活菌疫苗。pPMC5还可供构建预防牛、羊和人的痢疾活菌疫苗菌株用。