Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

The putative Orf4 of broad-host-range conjugative plasmid R446 could Be related to the H-NS family of bacterial nucleoid-associated proteins

The iml determinant of IncM plasmid R446 was described initially as a short fragment of DNA causing (when cloned in multicopy plasmids) insensitivity to pilus-dependent bacteriophage lysis in bacteria harboring coresident IncM plasmids. We have performed a computational analysis of the iml determinant of IncM plasmid R446 and found that a potential polypeptide (Orf4) shows similarity to the H-NS family of nucleoid-associated modulators of gene expression. The predicted protein has a molecular weight of 16.3 kDa and an isoelectric point of 5.50. Orf4 is adjacent and upstream of Orf5, a potential polypeptide related to the Hha/YmoA/RmoA family of proteins which modulate bacterial virulence expression (Hha and YmoA) and R100 transfer (RmoA) in response to environmental conditions.     

Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance

Introductionbla_OXA-48, bla_NDM-1 and bla_CTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described bla_OXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the bla_SHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania).MethodsSequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank.ResultsComparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the bla_OXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible.ConclusionThis work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.     

New R-plasmids in strains of Serratia marcescens with multiple drug resistance

In 4 S. marcescens polyresistant strains isolated from patients conjugative plasmids transferred to Escherichia coli have been detected. Two of these strains carry each one plasmid which codes resistance to 10 different antibiotics, including aminoglycosides which rarely occur in our country, and belongs to group IncC. The third strain is the host of 2 plasmids. One of them is similar to the above-mentioned 2 plasmids with respect to the incompatibility group and a set of markers, but additionally codes resistance to cephalosporins; the second plasmid has been determined as belonging to group IncM, unstable and capable of rendering the cells highly resistant only to aminoglycosides. And, finally, the fourth strain also carries 2 plasmids: one of them is unstable and belongs, supposedly, to group IncI alpha, and the second plasmid is stable and belongs to group IncM. The plasmid of group IncI alpha differs from all other plasmids of our Serratia by its capacity of rendering the cells highly resistant to chloramphenicol.     

Distribution of transferable trimethoprim and gentamicin resistance plasmids in Enterobacteriaceae

Following the employment of trimethoprim/sulfonamid and gentamicin in the general clinical praxis transferable plasmids with respective resistance function were identified at first in enteric bacteria from sewage before they could be detected in patient strains. Whereas the trimethoprim resistance plasmids represent different incompatibility groups (C, FII, I5, K, M, N, S, U) the gentamicin resistance plasmids are very similar IncM members of 62 MD in size. Therefore a clonal distribution of a particular gentamicin resistance plasmid has to be taken into consideration.     

Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12

Representative plasmids for most incompatibility groups in Escherichia coli K-12 were transferred to a "bald" strain to compare transfer frequencies for liquid and solid media. Standard broth matings were used for a liquid environment, but for solid surface mating, conjugation was allowed to take place on nutrient plates before washing off the cells for transconjugant selection on plates containing appropriate drugs. Plasmids that determine rigid pili transferred at least 2,000x better on plates than in broth. Some plasmids that determine thick flexible pili transferred 45 to 470x better, whereas others transferred equally well in both environments, as did plasmids of the I complex, which determine thin flexible pili. These results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively. The temperature-independent IncH2 plasmid R831b transferred best on plates, but other IncH plasmids transferred equally well in broth. This inconsistency led to the reclassification of R831b as IncM.     

Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates

We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, SalI, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactams, chloramphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occurred in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype.     

Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b

Tn7 insertion mutagenesis has been used to facilitate the generation of a physical (restriction endonuclease) and genetic map of the IncM plasmid, R831b. The only selectable phenotypes carried by this 90-kb conjugative plasmid are resistances to inorganic mercury [Hg(II)] and to organomercury compounds. Mutants in the Hgr locus of R831b complemented previously described mutants in the mer operon of the IncFII plasmid R100, indicating functional homology of the locus in each of these different plasmids. However, the R831b Hgr locus is not notably similar in restriction site pattern to either the mer operon of R100 or the mercury resistance transposon, Tn501. Although the enzymes they encode are co-ordinately regulated, the Omr locus of R831b maps approx. 13.5 kb away from the Hgr locus. Three insertions which affect neither phenotype lie between the Hgr and Omr loci; thus, the loci are separated both physically and genetically. One mutant was obtained which tentatively identifies the position of the Tra locus of R831b as adjacent to the Hgr locus.     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genesable to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.     

In vivo formation of a plasmid cointegrate expressing two incompatibility phenotypes

Plasmids of the N- and M-incompatibility groups were identified in a clinical isolate of Shigella sonnei. The IncN plasmid pDT200 encoded resistance to trimethoprim, streptomycin, and sulfamethoxazole, and specified N-type pili which were synthesized constitutively. The IncM plasmid mediated resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. It was fi⁺ and was repressed for pilus synthesis. Pili specified appeared to be of the FII-type. A recombinant plasmid was formed on transfer of these plasmids from Sh. sonnei to Escherichia coli. The cointegrate plasmid expressed two incompatibility phenotypes and encoded two types of pilus. The incompatibility behavior of the cointegrate plasmid formed in vivo resembled that of composite plasmids constructed by in vitro linkage experiments. The finding that plasmids from clinical isolates may express two incompatibility phenotypes and two types of pilus, and that the pilus type and incompatibility group may not necessarily correspond, has important implications for plasmid classification schemes.     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genes able to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Abstract Regulation of capsular biosynthesis ( rcs ) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA -dependent transfer of part of the Klebsiella chromosome to E. coli . Once resident in E. coli , the rcs gene(s) could not be mobilised to other strains of E. coli , and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli . These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli .     

Recovery of Cell-Bound Interferon

Interferon could be recovered from homologous cells to which it was applied but could not be recovered from heterologous cells. The amount of interferon that could be recovered from cells corresponded to the sensitivity of the cells to the antiviral activity of the interferon: mouse embryo fibroblasts, which were 5 to 10 times as sensitive as L-929 cells to interferon, bound 5 to 10 times more interferon than the latter, whereas Lpa cells, which were only one-third as sensitive as L-929 cells to interferon, bound only one-third as much as the latter. The concentration of cell-bound interferon was as much as 150 times the extracellular concentration of interferon applied to the cells. Interferon bound to cells at 4 C with the same efficiency as it did to cells at 37 C, and actinomycin D-treated cells bound interferon as well as normal cells. Even though the total amount of interferon bound to cells was as much as 30% of the amount of interferon applied to them, no loss of antiviral activity was detectable from the medium.     

农牧结合生态工程的基本理论与实践

农牧结合生态工程是一个复杂的农业生态、经济和技术系统工程,它由植物(种植业)、动物(养殖业)和微生物(连接种养业)3个子系统组成,其关键是建设一个以"四、三、二、一"畜牧业生产模式为主要内容的高效节粮型畜牧生态工程,它要求用尽可能少的自然资源,在尽可能短的周期内,生产尽可能多而优的农(牧)产品,以获取尽可能高的经济效益,达到或维持尽可能最佳的生态平衡.     

Tissue microarrays as a platform for proteomic investigation

Tissue microarrays have become an essential tool in translational pathology. They are used to confirm results from other experimental platforms, such as expression microarrays, as well as a primary tool to explore the expression profile of proteins by immunohistochemical analysis. Tissue microarrays are routinely used molecular epidemiology, drug development and determining the diagnostic, prognostic and predictive value of new biomarkers. By applying traditional protein based assays, as well as novel assays to the platform, tissue microarrays have gained a new utility as a proteomic tool for both basic science as well as clinical investigation. This article will explore the new approaches that are being applied to tissue microarrays to, characterize the human proteome, and new technologies that allow tissue microarrays to function as a protein array. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Wanted,an Anthrax vaccine: Dead or Alive?

It has been more than 100 years since the realization that microbes are capable of causing disease. In that time, we have learned a great deal as to how each organism has adapted to the immune system so as to avoid elimination. As well, we have also learned an immense amount since Louis Pasteur first proposed that the solution to infectious diseases was to culture the microbes and attenuate their virulence, so as to use them as vaccines. From the optimism and promise of the 19th century and immunization as the ultimate answer to the invasion by the microbial world, to the scientific realities of the 21st century, it is of interest to retrace the steps of the earliest microbiologists cum immunologists, to realize how far we've come, as well as how far we yet have to go. This editorial focuses on the history of anthrax as a microbial disease, and the earliest efforts at producing a vaccine for its prevention.     

Variability in Race Tests with Heterodera glycines

Tests of Heterodera glycines on differential host plants to determine races were run in Arkansas, Illinois, and North Carolina to check the uniformity of results of the test. Methods used at the three locations varied somewhat. Results indicate that the race test is highly variable. Isolates previously identified as race 1 were identified as race 1 or race 3; those identified as race 2 were identified in these tests as race 2, 4, 9, or 14; those previously identified as race 3 were identified as race 1 or race 3; those identified as race 4 were identified in these tests as race 4 or race 14; those previously identified as race 5 were identified as race 2; and those previously identified as race 6 were identified as race 1, 2, 4, 5, or 6. Part of the variability resulted from the use of differential host plants from different sources and part from nonstandard differential host plants. Other variations may be due to inability to obtain completely uniform inoculum or to recover all nematodes that penetrated.     

Industrial and biotechnological applications of laccases: a review

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.     

SLX4: multitasking to maintain genome stability

The SLX4/FANCP tumor suppressor has emerged as a key player in the maintenance of genome stability, making pivotal contributions to the repair of interstrand cross-links, homologous recombination, and in response to replication stress genome-wide as well as at specific loci such as common fragile sites and telomeres. SLX4 does so in part by acting as a scaffold that controls and coordinates the XPF–ERCC1, MUS81–EME1, and SLX1 structure-specific endonucleases in different DNA repair and recombination mechanisms. It also interacts with other important DNA repair and cell cycle control factors including MSH2, PLK1, TRF2, and TOPBP1 as well as with ubiquitin and SUMO. This review aims at providing an up-to-date and comprehensive view on the key functions that SLX4 fulfills to maintain genome stability as well as to highlight and discuss areas of uncertainty and emerging concepts.     

Synthesis of 4-cyanophenyl and 4-nitrophenyl 1,5-dithio-D-ribopyranosides as well as their 2-deoxy and 2,3-dideoxy derivatives possessing antithrombotic activity

1,2,3,4-Tetra-O-acetyl-5-thio-D-ribopyranose as well as its 1-bromide were used as donors in the reaction with 4-cyano- and 4-nitrobenzenethiol, to give the corresponding thioglycosides in different anomeric ratios depending on the reaction conditions. Zemplén deacetylation afforded 4-cyanophenyl as well as 4-nitrophenyl 1,5-dithio-alpha- and beta-D-ribopyranosides, respectively. 1,3,4-Tri-O-acetyl-2-deoxy-5-thio-D-erythro-pentopyranose was synthesized from methyl 2-deoxy-D-erythro-pentofuranoside and was coupled with 4-cyano- and 4-nitrobenzenethiol to give anomeric mixtures from which 4-cyanophenyl as well as 4-nitrophenyl 1,5-dithio-beta-D-erythro-pentopyranosides were isolated after deacetylation. 1,4-Di-O-acetyl-2,3-dideoxy-5-thio-D-glycero-pentopyranose was obtained starting from 1,2,5,6-di-O-isopropylidene-D-mannitol and used as the donor in the glycosylation reaction with 4-cyano- and 4-nitrobenzenethiol. The resulting anomeric mixtures were separated to give, after deacetylation, 4-cyanophenyl as well as 4-nitrophenyl 2,3-dideoxy-1,5-dithio-beta-D-glycero-pentopyranosides. All of these thioglycosides showed significant antithrombotic activity on rats after oral administration.