Sa/I restriction endonuclease maps of FII incompatibility group R plasmids.

SalI restriction endonuclease maps of FII incompatibility group R plasmids NR1, NR84, and R6 have been determined by sequential digestion of plasmid DNA with EcoRI and SalI and subsequent analysis of the fragments by electrophoresis on agarose gels. In the composite R plasmid NR1 there are five SalI sites, one in the r-determinants component and four in the RTF-Tc component. SalI cleavage of transitioned NR1 DNA, which contains tandem sequences of the r-determinants in a head-to-tail orientation, produces the five original bands plus a single new amplified band whose mobility on agarose gels corresponds to the monomer r-determinants DNA. NR84 has a total of four SalI sites. It is missing one of the SalI sites near the repA locus. R6 has five SalI sites, four the same as those of NR84, and one additional site within the Km transposon Tn601.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Molecular genetic study of the changes in the incompatibility and regulation of the transfer of the F-like pAP18-1 plasmid induced by nitrosoguanidine and the Tn5 and Tn9 transposons

Relation between induced mutations of plasmid pAP18-1 (Tc, Col) and alterations in it's restriction map was studied. Nitrosoguanidine induced mutations of transfer regulation system and incompatibility of this plasmid related with alteration in the situation of recognition sites for restrictases EcoR1 and Sal1 in map positions 42.2-4.3 and 12.9-17.9 MD. Insertions of transposons Tn5 and Tn9 into the plasmid DNA resulted in a decrease of incompatibility level.     

Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1.

A 1.45-megadalton segment of DNA cloned from a miniplasmid derived in vivo from a copy number mutant of the R plasmid NR1 has been shown to contain all functions essential for incompatibility and autonomous plasmid replication in Escherichia coli. Specific endonuclease cleavage sites within this DNA segment that localize functions required for replication have been mapped. A 0.45-megadalton fragment that specifies the FII incompatibility of NR1 has been identified within the replication region, and DNA fragments containing this incompatibility region, but lacking other functions required for replication, have been cloned.     

Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.     

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

Identification of Potential Small Molecule Allosteric Modulator Sites on IL-1R1 Ectodomain Using Accelerated Conformational Sampling Method

The interleukin-1 receptor (IL-1R) is the founding member of the interleukin 1 receptor family which activates innate immune response by its binding to cytokines. Reports showed dysregulation of cytokine production leads to aberrant immune cells activation which contributes to auto-inflammatory disorders and diseases. Current therapeutic strategies focus on utilizing antibodies or chimeric cytokine biologics. The large protein-protein interaction interface between cytokine receptor and cytokine poses a challenge in identifying binding sites for small molecule inhibitor development. Based on the significant conformational change of IL-1R type 1 (IL-1R1) ectodomain upon binding to different ligands observed in crystal structures, we hypothesized that transient small molecule binding sites may exist when IL-1R1 undergoes conformational transition and thus suitable for inhibitor development. Here, we employed accelerated molecular dynamics (MD) simulation to efficiently sample conformational space of IL-1R1 ectodomain. Representative IL-1R1 ectodomain conformations determined from the hierarchy cluster analysis were analyzed by the SiteMap program which leads to identify small molecule binding sites at the protein-protein interaction interface and allosteric modulator locations. The cosolvent mapping analysis using phenol as the probe molecule further confirms the allosteric modulator site as a binding hotspot. Eight highest ranked fragment molecules identified from in silico screening at the modulator site were evaluated by MD simulations. Four of them restricted the IL-1R1 dynamical motion to inactive conformational space. The strategy from this study, subject to in vitro experimental validation, can be useful to identify small molecule compounds targeting the allosteric modulator sites of IL-1R and prevent IL-1R from binding to cytokine by trapping IL-1R in inactive conformations.     

Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites

Protein kinase C (PKC) phosphorylates the NR1 and NR2A subunits of NMDARs at consensus sites located within their intracellular C-terminal tails. However, the functional consequences of these biochemical events are not well understood. In HEK293 cells expressing NR1/NR2A, activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate (PMA) increased NMDAR desensitization as evidenced by a reduced steady-state current without any change in peak. The effects of PMA on NMDAR-mediated responses were prevented by specific PKC inhibitors and were not mimicked by an inactive enantiomer of PMA. The effects of PMA were preserved despite mutagenesis of the major PKC sites on the NR1 subunit (S889A, S890A, S896A and S897A) or removal of the entire NR1 C-terminal tail (NR1(stop838)). When co-expressing NR1(stop838)/NR2A the effects of PMA could only be observed with agonist concentrations sufficient to induce glycine-insensitive desensitization. Moreover, the effects of PMA were observed in receptors composed of NR1/NR2A and NR1/NR2B, but not NR1/NR2C, a subunit combination in which desensitization is absent. The NR2 subunit dependence suggested that the actions of PMA might require specific PKC sites previously identified within NR2A. However, a C-terminal truncated form of NR2A (NR2A(stop905)) remained responsive to PMA. We conclude that activation of PKC increases NMDAR glycine-insensitive desensitization independently of previously identified sites located within the NR1 C-terminus and distal segment of the NR2A C-terminus.     

Characteristics of Klebsiella pneumoniae plasmid bearing the markers of drug resistance and antilysozyme activity

Electrophoretic study of the profile of plasmid DNA in agarose gel has shown the presence of a plasmid with a molecular weight of 55-60 MD in K. pneumoniae strains possessing antilysozyme activity. Plasmid pAlz60 of K. pneumoniae 22-110, isolated from the blood of a septicemia patient, is a fi- type conjugative plasmid. This plasmid is transferred to recipient strains of different species of enterobacteria with a frequency of 1 X 10(-5) to 1 X 10(-7). Simultaneously with the transfer of the plasmid, recipient cells inherit the antilysozyme markers and resistance to a number of drugs. The discovered plasmid has one restriction site for each of endonucleases EcoRI and XhoI and 16-20 sites for restrictases KpNI, BglII and Hind III.     

Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.     

Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors

The impact of two plasmid (47, 82 MD), single plasmid (47 MD) and non plasmid Y. pseudotuberculosis strains, Y. enterocolitica (47 MD) as well as Y. pseudotuberculosis superantigen (YPM) on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN = alpha) and tumor necrosis factor alpha (TNF-alpha) by whole blood cells obtained from donors was studied. All Y. pseudotuberculosis and Y. enterocolitica strains stimulated the production of IFN-alpha, IL-1, IL-6 and TNF-alpha by whole blood cells, but considerably less than Y. pseudotuberculosis lipopolysaccharide and YPM. These data are indicative of the pathogenetic role played by 82 MD plasmid in manifestation of Y. pseudotuberculosis immunosuppressive properties. The maximum stimulation of the production of cytokines was observed under the action of YPM, which confirmed an important role played by this superantigen in the pathogenesis of Y. pseudotuberculosis.     

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.     

Conjugative R plasmids isolated from hospital strains of Pseudomonas aeruginosa]

It was shown that Pseudomonas aeruginosa hospital strains isolated from patients and environment in the Republican Centre of Burns in Tbilisi contained conjugative R plasmids. The plasmids were marked pM15 and pM19, respectively. The plasmid pM15 determined resistance to carbenicillin, kanamycin and tetracycline and plasmid pM19 determined resistance to carbenicillin, kanamycin, tetracycline, chloramphenicol, gentamicin and streptomycin. Plasmid pM15 had a molecular weight of 45.8 MD and seven sites for EcoRI, six sites for HindIII and five sites for Hpa-I-restrictase. This plasmid, as others, belongs to the Inc-P1 incompatibility group.     

地中海拟无枝菌酸菌U-32硝酸还原酶的基因克隆

Southern杂交分析表明在地中海拟无枝菌酸菌U-32染色体DNA和黑曲霉niaD(硝酸还原酶基因)之间存在着明显的同源性。利用异源niaD探针从地中海拟无枝菌酸菌U-32基因文库中筛选得到一个能与niaD杂交的5.0kb的PstⅠ片段。该片段经同位素标记后能与地中海拟无枝菌酸菌U-32染色体上一个相同的PstⅠ片段杂交,位于这一片段上的2.1kb SmaⅠ-EcoR Ⅴ片段只能与以硝酸盐为唯一氮源的总RNA杂交,而不能与相同条件下以铵盐为唯一氮源的总RNA杂交,这些结果表明,所克隆到的5.0kb PstⅠDNA片段含有地中海拟无枝菌酸菌U-32的硝酸还原酶基因。这是好氧细菌硝酸还原酶基因克隆的首次报道。由该酶蛋白分子量推测,其结构基因大小在1.5kb左右,进一步的杂交分析发现在5.0kb的PstⅠ片段中含有完整的NR基因。用20种限制酶对重组质粒pJL1进行了限制酶酶谱的构建,发现有10种酶在pJL1外源片段上无切点,6种酶为单切点,EcoRⅠ与SmaⅠ各有两个切点。     

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.     

Functional analysis of replication and stability regions of broad-host-range conjugative plasmid CTX-M3 from the IncL/M incompatibility group

Plasmid CTX-M3 (89 kb) isolated from Citrobacter freundii from a Warsaw hospital is a mosaic plasmid with replication functions 100% identical with those of pMU407.1 of the IncL/M group, conjugative operons with up to 60% homology to ColIb-P9 (IncI) and stability functions originating either from NR1(R100) (IncFII) or ColIb-P9 /R1/NR1 plasmids. We established the broad-host-range for pCTX-M3 and defined its minireplicon in Escherichia coli. We analyzed the role of stability cassettes and showed that the par operon consists of three orfs parA (stbA), parB (stbB) and nuc with a centromere-like region located upstream of the operon. Deletion of the par operon strongly destabilized pCTX-M3 despite the presence of the pemIK toxin-antidote system identical to that on NR1(R100) plasmids. Deletion of the pemIK operon had no effect on plasmid stability.