Survival of genetically engineered Escherichia coli in natural soil and river water

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased. and accepted 19 August 1989     

Survival of genetically engineered Escherichia coli in natural soil and river water

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased.     

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.     

Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction map of E. coli shuttle plasmid (p# GTE5) with secretive function

The shuttle plasmid (p# GTE5) DNA with secretive function was extracted by the alkali lysozyme method from E. coli RRI strain. Its molecular weight is 4.5 Md and DNA size is 6.9 Kb. Restriction fragments of plasmid was obtained by single and double enzymes complete digestion using five different restriction endonucleases. The restriction map of shuttle plasmid (p# GTE5) was established for the enzymes EcoRI, BglII, pstI, PvuII, and TaqI.     

类杆菌5-硝基咪唑抗性质粒pIP419的研究

5-硝基咪唑类药物广泛应用于厌氧菌感染症的临床治疗,近几年厌氧菌5-硝基咪唑抗性菌株的报道有所增加。本文介绍分离自类杆菌的编码抗5-硝基咪唑抗性质粒pIP419的研究结果。pIP419分子量为10kb,已建立了限制性内切酶物理图谱。该质粒可通过接合和转化在不同的菌种或菌株间转移。质粒上负责质粒复制和转移的基因片段已被定位和克隆。     

Hybrid vectors for the cyanobacterium Anacystis nidulans R2, containing the plasmid pUB110 from Staphylococcus aureus

The recombinant vector plasmids were constructed having the DNA of pUB110 plasmid (4,5 kb, KmR) from Staphylococcus aureus inserted into the cryptic plasmids pANS (8 Kb) and pANL (48,5 kb) of cyanobacterium Anacystis nidulans R2. The hybrid plasmids transform cyanobacterial cells to Km-resistance with high efficiency. The plasmid pBS20, containing the complete sequence of pANS and pUB110 DNA, transforms Bacillus subtilis rec E4 protoplasts being, however, unstable in bacilli cells and disintegrates deriving a parent pUB110 plasmid.     

Characterization of large plasmids encoding resistance to toxic heavy metals in Salmonella abortus equi

Salmonella abortus equi vaccine strains were found to be resistant to high levels of toxic heavy metals--arsenic, chromium, cadmium, and mercury. The two strains 157 and 158 were resistant to ampicillin also. Curing of these strains resulted in loss of one or more resistance marker indicating plasmid borne resistance. Plasmid profile of strain 157 showed presence of three plasmids of 85, 54, and 0.1 Kb, whereas 158 strain showed presence of 85 Kb and 2 Kb plasmids. Plasmids were isolated from strain 157 and introduced into E. coli DH5alpha with a transformation efficiency of 2 x 10(3) transformants/microg DNA. Interestingly the transformants were resistant to antibiotics, heavy metals (As, Cr, Cd, Hg) and was also able to utilize citrate, a trait specific to Salmonella species. We report and establish for the first time the transferable large plasmids encoding resistance to various heavy metals, antibiotics and biochemical nature of S. abortus equi.     

Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR

The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7. These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR. Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205. The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene.     

Sequencing and Genetic Variation of Multidrug Resistance Plasmids in Klebsiella pneumoniae

Background

The development of multidrug resistance is a major problem in the treatment of pathogenic microorganisms by distinct antimicrobial agents. Characterizing the genetic variation among plasmids from different bacterial species or strains is a key step towards understanding the mechanism of virulence and their evolution.

Results

We applied a deep sequencing approach to 206 clinical strains of Klebsiella pneumoniae collected from 2002 to 2008 to understand the genetic variation of multidrug resistance plasmids, and to reveal the dynamic change of drug resistance over time. First, we sequenced three plasmids (70 Kb, 94 Kb, and 147 Kb) from a clonal strain of K. pneumoniae using Sanger sequencing. Using the Illumina sequencing technology, we obtained more than 17 million of short reads from two pooled plasmid samples. We mapped these short reads to the three reference plasmid sequences, and identified a large number of single nucleotide polymorphisms (SNPs) in these pooled plasmids. Many of these SNPs are present in drug-resistance genes. We also found that a significant fraction of short reads could not be mapped to the reference sequences, indicating a high degree of genetic variation among the collection of K. pneumoniae isolates. Moreover, we identified that plasmid conjugative transfer genes and antibiotic resistance genes are more likely to suffer from positive selection, as indicated by the elevated rates of nonsynonymous substitution.

Conclusion

These data represent the first large-scale study of genetic variation in multidrug resistance plasmids and provide insight into the mechanisms of plasmid diversification and the genetic basis of antibiotic resistance.     

Structural-functional organization of R-plasmid pBS222 with a broad range of bacterial hosts

The broad host-range plasmid pBS222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pBS222 is efficiently mobilized by Inc P-1 plasmid RP4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. The size of the plasmid (17.2 Kb) has been determined and its physical map has been constructed. The plasmid harbours the unique sites for restriction endonucleases BglII, HindIII, HpaI, KpnI, SmaI and XbaI cleawage. The plasmid derivatives pBS352-pBS355 have been obtained that carry kan- and cam-determinants in addition to tet-gene. Plasmid pBS355 has been used to clone EcoRI-fragments of phage lambda DNA. The plasmid pBS222 regions essential for replication and maintenance have been localized by DNA hybridization analysis of its mini-derivatives pBS356 and 357. pBS222 is a convenient model for investigations of the plasmid replication and maintenance mechanisms in different bacterial hosts as well as for the construction of broad host-range vectors.     

A new type of plasmid from a wild isolate of Dictyostelium species: the existence of closely situated long inverted repeats.

A circular plasmid having high copy number was found in a wild isolate of Dictyostelium species. Gel electrophoresis, electron microscopy and Southern blot hybridization revealed that the plasmid, named pDG1, is 4.5 Kb (1.5 micron) in size with closely situated long inverted repeats. The plasmid seems to be located in the nuclei. It was not a derivative of ribosomal DNA. The possible correlation of the plasmid with the putative intermediate DNA of retrotransposon DIRS-1 found in Dictyostelium discoideum is discussed.     

Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IFO 3455

An 8.3 Kb DNA fragment was cloned from Pseudomonas aeruginosa IFO 3455. This fragment-containing Escherichia clone, pEL2, produced a high level of elastase activity. A smaller EcoRI-KpnI fragment was subcloned into pUC118 and E. coli HB101 was transformed with the plasmid. A deletion mutant clone was also constructed in the same bacteria. These deletion mutants were tested for elastase activity and it became clear that the full length of the elastase gene was 1.0-1.3 Kb. DNA sequencing analysis revealed that this DNA fragment contains the DNA sequence coding N-terminal amino acid sequence of the elastase protein.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.     

Analysis of somatic cell H-2 variants to define the structural requirements for class I antigen expression

We have taken the approach of producing somatic cell variants with altered H-2 products to study the structural requirements for cell surface expression of class I histocompatibility molecules. H-2 antigen variants generated by chemical mutagenesis of a cell line expressing the H-2b haplotype were first selected with alloantisera for their loss of H-2Kb expression, and then were analyzed by radioimmunoassay for the appearance of intracellular Kb antigen. For one such variant (69.9.15), whereas the H-2Kb antigen was absent from the cell surface as assayed by antibody-mediated complement-dependent cytotoxicity, an H-2Kb molecule was detected within the cell lysate as confirmed by direct immune precipitation with Kb-specific monoclonal antibodies. The product had an altered antigenic phenotype, since it reacted with only two anti-Kb monoclonal antibodies (Y-3 and EH-144) and not with a third (5F1.2). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the beta 2 microglobulin-associated, intracellular H-2Kb heavy chain to be slightly smaller in Mr than the H-2Kb of the parental cell line. Hybridization analysis revealed the Kb gene from the variant to be without gross alterations, and furthermore, identified a Kb mRNA species that was identical in size to wild-type Kb mRNA. Because complementation was not observed after somatic cell fusion of variant cells with BALB/c splenocytes, it appeared that the alteration in Kb expression was due to a cis-acting defect. In addition, DNA-mediated gene transfer of the wild-type Kb gene into the variant cell line resulted in expression of the Kb antigen on the cell surface, thus confirming that the defect in expression of the mutant Kb product was not due to other factors in the 69.9.15 cell line. Such findings are consistent with the conclusion that stable H-2Kb surface-negative somatic variants can arise due to limited alterations in the Kb gene, resulting in the synthesis of a class I molecule that is expressed only as an intracellular product.