H1 Plasmids Research Articles

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

An oligonucleotide microarray to characterize multidrug resistant plasmids

Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replicon type Inc A/C and Inc H1. It is important to understand the transmission of these MDR plasmids because the genes they carry can affect the outcome of antimicrobial therapy. The aim of this study was to design a microarray with oligonucleotide probes for every gene in the six Inc A/C and one Inc H1 plasmids of interest while representing all redundant sequences only once. The microarray is printed in triplicate with 493 unique oligonucleotide probes 70 nucleotides in length. Salmonella enterica and Escherichia coli control strains and test plasmids (in the parent strain and transformed into a known E. coli background strain) were hybridized to the plasmid microarray. This hybridization arrays presents a rapid and cost effective method for high-density screening of isolates to evaluate the gene content of Inc A/C and H1 plasmids and will show how plasmids can change content with transmission.

Molecular cloning and expression of theStreptococcus lactistagatose 1,6-bisphosphate aldolase gene inEscherichia coli

A 4.4 kb EcoRI DNA fragment of the Streptococcus lactis H1 plasmid pDI1 was cloned into the Escherichia coli plasmid pACYC 184. The recombinant plasmid expressed d-tagatose 1,6-bisphosphate aldolase activity in E. coli. Enzyme activity was at the same level as in the original S. lactis host but was not repressed by glucose.

Genetic and molecular characterization of conjugative R plasmids detected in Salmonella strains isolated from humans and feral pigeons in the same district.

A total of 24 conjugative R plasmids were isolated from 19 Salmonella typhimurium strains from human patients with acute diarrhea in Hiroshima City. Moreover, 6 R plasmids were detected in 4 S. typhimurium strains and 1 S. litchfield strain which were isolated from pigeon droppings collected at 3 parks of the city. Genetic properties of these R plasmids of human and pigeon origins were used as an epidemiologic marker to confirm the possibility of transmission of Salmonella between pigeons and humans. Incompatibility (Inc) group H1 (12 plasmids-50.0%) was most frequently detected among the 24 human plasmids, followed by IncIα (4-16.7%) and FII (1-4.2%). Three of the 6 plasmids of pigeon origin were classified into IncIα and 1 into H1. IncIα plasmids conferring resistance to streptomycin (Sm), sulfadimeth-oxine (Su) and tetracycline (Tc) with or without mercury resistance and IncH1 plasmids conferring resistance to chloramphenicol, kanamycin, Sm, Su and Tc with or without mercury resistance were detected in Salmonella strains of both human and pigeon origins. Half of the IncIα plasmids and all of the H1 plasmids described above were derived from parent S. typhimurium subserovar copenhagen strains with the same biovar, respectively. Five IncIα and two IncH1 R plasmids of human and pigeon origins were about 76.9 and 120 megadaltons in size, respectively, and the plasmid DNAs of each Inc group showed identical restriction pattern. On the basis of the distribution of the same R plasmids in particular or non-particular Salmonella strains from humans and pigeons, the possibility of association of pigeons with human salomonellosis was presumed.

Genetic studies of H group plasmids by bacteriophage P1 transduction.

Bacteriophage P1 transduction was used to study the incompatibility group H1 plasmid pRG1251, molecular weight 120 x 10(6), and the incompatibility group H2 plasmid pSD114, molecular weight 166 x 10(6). The order of resistance (R) determinants on pSD114 was deduced from transduction and segregation experiments to be chloramphenicol-tetracycline-kanamycin-streptomycin. Resistance to tellurium and to coliphages, which are properties also encoded by many H2 plasmids, were not transduced with the other markers. On pRG1251, the ampicillin and tetracycline resistance markers appear to be located together, as do the chloramphenicol, streptomycin, and sulfamethoxazole resistance markers. Frequently, blocks of R determinants were transposed to the P1 genome or to the Escherichia coli chromosome. P1 DNA was isolated which carried the chloramphenicol, streptomycin, and sulfamethoxazole markers from pRG1251 and had a molecular weight of 64 x 10(6). Other P1 prophages carried R determinants from pSD114 and had molecular weights of 86 x 10(6). A plasmid of molecular weight 124 x 10(6) was also isolated which contained incompatibility determinants from P1 (incompatibility group Y) and from the H2 group plasmid. The mechanism of formation of these unusual plasmid species is discussed.

PJT2: unusual H1 plasmid in a highly virulent lactose-positive and chloramphenicol-resistant Salmonella typhimurium strain from calves

A lactose-positive and chloramphenicol-resistant strain of Salmonella typhimurium of high virulence was isolated from an outbreak of enteric and septicemic salmonellosis in veal calves. the lactose-positive marker was located on an H1 plasmid, pJT2, together with resistance to chloramphenicol, streptomycin, sulfonamides, and tetracycline, pJT2 was unusually large and had a molecular weight of about 150 X 10(6). A similar plasmid was also present in a third of the Escherichia coli strains isolated from the intestines of septicemic calves during the outbreak. Spontaneously derived Lac- derivatives of pJT2 had an approximate molecular weight of 140 X 10(6).

Plasmids in Escherichia coli controlling citrate-utilizing ability

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.

Thermosensitive H1 Plasmids Determining Citrate Utilization

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).

Thermosensitive antibiotic resistance plasmids in enterobacteria.

Of 775 conjugative plasmids found in enterobacteria mediating antibiotic resistance, 24 (3.1%) were thermosensitive (ts); they were most common in Klebsiella pneumoniae. Ts plasmids were also found in all the samples of sewage and river water examined. Over half of 73 ts plasmids from unrelated sources mediated resistance to chloramphenicol in addition to several other antibiotics. Many of them mediated resistance to mercury (53.4%), arsenite (38.4%) and tellurite (79.5%) but not to copper, cobalt and silver. Fifty-eight belonged to incompatibility group H2 and 12 belonged to the H1 group. Resistance to mercury, arsenite and tellurite was common in strains containing H2 plasmids but not in H1 plasmids. The 73 plasmids transferred at high rates at 22 and 28 degrees C and at lower rates at 15 degrees C; they transferred at very low rates or not at all at 37 degrees C. They could be divided into two sets according to whether they transferred at a high or at a low rate at 33 degrees C. Unlike the prototype plasmid, Rts 1, they were solely or mainly ts for transfer and not for replication and only one of them brought about a marked reduction in growth rate of its host organism at 42 degrees C. None of the 73 plasmids mediated colicin or haemolysin production. Three plasmids, all from K. pneumoniae, mediated utilization of lactose, two of sucrose and raffinose and three, all belonging to group H1, of citrate. None of the plasmids increased the pathogenicity of Salmonella typhimurium for chicks or Escherichia coli K12 for mice.

Incompatibility and surface exclusion properties of H1 and H2 plasmids

Plasmids of the H incompatibility group showed two types of surface exclusion and incompatibility interactions. Strong incompatibility and surface exclusion were evident between plasmids within the same subgroup, and recombination frequently occurred between these plasmids after antibiotic selection for the presence of two plasmids in the same cell. Weaker interactions were seen between plasmids of the different subgroups, H1 and H2, and recombination was not detected. Incompatibility between H1 and H2 plasmids led preferentially to the loss of the H1 plasmid, irrespective of the order of entry of the plasmids. These data are consistent with the hypothesis that incompatibility is negatively controlled.

Incompatibility and bacteriophage inhibition properties of N-1, a plasmid belonging to the H2 incompatibility group

N-1, a plasmid isolated from a strain of Shigella flexneri in Japan more than 10 years ago, mediates the phage inhibition phenotype which has recently been found to be characteristic of plasmids of the H2 incompatibility group. Using the criteria of phage inhibition, surface exclusion and incompatibility, the N-1 plasmid is shown to be closely related to H2 plasmids isolated from non-typhoid salmonella and distantly related to H1 plasmids isolated from Salmonella typhi. Plasmids of other incompatibility groups did not show the H2 type of phage inhibition.