Transition of the R Factor R12 in Proteus mirabilis

When Proteus mirabilis harboring the R factor R12 (a round of replication mutant of the R factor NR1) is cultured in medium containing streptomycin there can be an amplification in the number of copies of r-determinants per cell and the formation of enlarged polygenic R factors containing repeated sequences of r-determinants as well as polygenic molecules consisting of repeated sequences of r-determinants. This phenomenon has been referred to as the "transition." When transitioned cells are then cultured in drug-free medium, within a few generations two distinct density species of R factor deoxyribonucleic acid (DNA) are observed in a CsCl density gradient: a 1.712 g/ml band of covalently closed circular R factor DNA consisting of one transfer factor (RTF-TC) plus one r-determinant and a 1.718 g/ml band consisting of repeated sequences of r-determinants. The RTF-TC component of the R factor appears to control the replication of all the R factor DNA which is attached to it. In the autonomous state, however, polygenic sequences of r-determinants do not appear to replicate under the same control mechanism as when they are attached to an RTF-TC.     

R Factor Transmission In Vivo

Experimental infections were induced in weanling pigs orally both with nalidixic acid (NA)-sensitive and -resistant strains of Salmonella choleraesuis var. kunzendorf, designated RC221 and RC221NA, respectively. Prior to the time of infection, cultures of normal bacterial flora were isolated from swine fecal matter and screened for the presence of R factors. A majority of these bacterial isolates harbored transferable resistances. Both strains RC221 and RC221NA have been shown to be competent recipients in vitro of the R factors present in the normal intestinal flora. The property of NA resistance greatly facilitated recovery of the infecting organism. After infection, salmonellae from liver, lung, spleen, lymph node, intestine, and feces were screened for the presence of R factors. Transfer of drug resistance in vivo was a rare occurrence; however, if infected specimens, particularly intestinal, were incubated in nutrient broth prior to plating, R factor transfer occurred, presumably in the test tube. Changes in recipient cultures were frequently observed after introduction of R factors from organisms of pig origin into the S. choleraesuis var. kunzendorf test organisms. Alterations include changes in typing reaction, granular growth in broth, differences in colony form, and reduction of virulence.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Membrane-Associated R Factor Proteins

R factor proteins are synthesized in R factor-containing Escherichia coli minicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Impaired Transduction of R213 and Its Recovery by a Homologous Resident R Factor

Transduction by Plkc of drug-resistance markers of the factor R213 was shown to occur at an exceptionally low frequency (at less than 10(-8) of the input phage), and they could not be transduced by P22. When the recipient cells carried a homologous R factor derived from R213, markers were transduced by Plkc at a normal frequency (at about 10(-5) to 10(-6) of the input phage). Derivative R factors, transducible by Plkc at a normal frequency but being transferred by conjugation at a frequency lower than that of the original R213, were obtained. This type of transductant often segregated R(-) cells. In addition, several transductants contained R factors which were transferred normally by conjugation but were transduced by Plkc at as low a frequency as the original R213. This type of transductant was an effective recipient for transduction by Plkc of R213 when apparently "cured" by acridine treatment. No such effective "cured" recipients were obtained from the transductants with derivatives of R213 transducible at a normal frequency. Two possible interpretations are presented: (i) R213 produces a bacteriocin-like substance upon transduction, or (ii) the genome size of R213 is too large for all of its determinants to be transduced.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.     

Composite Circular Forms of R Factor Deoxyribonucleic Acid Molecules

Two R factors, one (R15) conferring resistance to streptomycin and sulfonamide (SM(r)SU(r)) and the other (222/R3) to streptomycin, sulfonamide, and chloramphenicol (SM(r)SU(r)CM(r)), were transferred to a Proteus mirabilis strain, and deoxyribonucleic acid (DNA) extracted from these strains was subjected to density-gradient centrifugation. R15-DNA formed a single satellite band at a density of 1.709 g cm(-3). Electron microscopy of samples from this band showed circular molecules of one type, with a contour length of 18 mum (35 x 10(6) daltons). 222/R3-DNA formed a satellite band with three peaks at densities 1.708, 1.711 and 1.717 g cm(-3). Electron micrographs revealed circular structures from each band with contour lengths, respectively, of 29 (54 x 10(6) daltons), 36 (68 x 10(6) daltons), and 6 mum (12 x 10(6) daltons). "Supertwisted" forms of several molecular species were found. It is suggested that 222/R3 DNA comprises either a single 36-mum molecule or two individual molecules, 29 and 6 mum in length, and that this may reflect the evolutionary development of R factors.     

Molecular Studies of R Factor Compatibility Groups

Molecular studies of R factors of six groups, F_II, I₁, I₂, N, B, and H, defined on the basis of compatibility, support the conclusions drawn from genetic studies. In general, R factors of a given compatibility group are similar in size. Deoxyribonucleic acid (DNA) reassociation occurs freely between members of the same group but is minimal between heterologous groups. An exception to this was found in group H, of which one factor showed minimal homology with the remaining plasmids of the group. A further exception was found with groups I₁ and B, which, although genetically distinct, show between 18 and 28% of DNA homology. Groups I₁ and I₂ are molecularly distinct, despite the fact that they both stimulate the synthesis of I-fimbriae.     

Isolation of Catenated Forms of R Factor DNA from Minicells

STUDIES^1–6 of the molecular nature of antibiotic resistance (R) factors in Escherichia coli have shown that several of them consist of covalently closed molecules of circular DNA. Use was made of this property in the separation of the R factor from chromosomal DNA of E. coli, which has a similar nucleotide-base composition. In selecting for covalently circular R factor DNA molecules, however, the procedures used in these earlier experiments-caesium chloride-ethidium bromide centrifuga-tion⁷ and bulk nitrocellulose adsorption²—necessarily selected against isolation of other (non-circular) forms of R factor DNA that might have been present.     

Properties of an R Factor from Pseudomonas aeruginosa

An R factor from Pseudomonas aeruginosa, which confers resistance to penicillins, kanamycin, and tetracycline, was studied in Escherichia coli K-12. The R factor could coexist with F-like or I-like plasmids and therefore constituted a novel compatibility group. The R factor was transferable from E. coli to bacterial genera outside the Enterobacteriaceae (Pseudomonas and members of the Rhizobiaceae) to which transfer of F-like and I-like plasmids could not be demonstrated.     

Relation of R Factor and Chromosomal β-Lactamase with the Periplasmic Space

The release of several R factor and chromosomal beta-lactamases by osmotic shock treatment was studied. It was found that those beta-lactamases with a molecular weight of about 20,000 were released, but those with a molecular weight of about 30,000 to 44,000 were not released during osmotic shock. This differential release did not depend on whether the structural genes were on the chromosome or on the genome of an R factor. The release or retention of the beta-lactamases appeared to be a characteristic of the enzyme rather than the host cell since the same results were obtained when the R factors were harbored by a variety of host bacteria. Studies with bacteria which produced more than one beta-lactamase showed that each enzyme reacted independently to the presence of other beta-lactamases produced by the host bacterium.     

Naturally Occurring R Factor, Derepressed for Pilus Synthesis, Belonging to the Same Compatibility Group as the Sex Factor F of Escherichia coli K-12

A naturally occurring R factor with constitutive pilus synthesis is described which resembles the sex factor F in compatibility and in restricting coliphage T7. Unlike F, it is not cured during growth with acridine orange. Results suggest that the R factor produces repressor of pilus synthesis, to which the operator is insensitive (i(+)o(c)). In this respect it differs from both the F factor (i(-)o(+)) and wild-type F-like R factors (i(+)o(+)).     

R Factor Deoxyribonucleic Acid in Chromosomeless Progeny of Escherichia coli

The deoxyribonucleic acid (DNA) of resistance (R) factor 222 carried by Escherichia coli strain P678-54 was found in the normally chromosomeless progeny (minicells) of that strain. The entry of the R222 DNA into minicells appears to be via segregation at the time of their formation from normal cells. The R222 DNA can replicate in minicells although the extent of its replication appears to be limited. An analysis of the R222 DNA structure indicates that it exists in minicells as double-stranded linear, open circular, and twisted circular monomers (molecular weight, about 6.2 x 10(7) daltons). The monomers visualized by electron microscopy are 31.0 +/- 0.5 mum in length. An examination of the effect of acridine orange on the replication of R222 and colicin E1 DNA indicates the dye intereferes with plasmid DNA replication.     

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF6 (EFA6) R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, pleckstrin homology (PH), and coiled coil (CC) domains of the other EFA6 family GEFs. Here we report the functional characterization of EFA6R. Endogenous EFA6R was present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R were targeted to the PM. In vitro, GFP-EFA6R associated weakly but preferentially with phosphatidylinositol 4,5-bisphosphate (PIP₂) through the PH domain. EFA6R required both its PH and CC domains localized at the C terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6RΔCC) was released from the PM into the cytosol upon PIP₂ depletion, whereas EFA6R release from the PM required both PIP₂ depletion and actin destabilization. These results suggest that the dual targeting via the PH and CC domains is important for the PM localization of EFA6R. EFA6R specifically catalyzed the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulated ARF6 localization and thereby actin stress fiber loss. The GEF activity of EFA6R was dependent on the presence of the Sec7 domain. The PH and CC domains were also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Pseudo-fi+ I-Like Sex Factor, R62(I), Selective for Increased Pilus Synthesis

An I-like R factor, R62(I), inhibits F-like pilus synthesis and selects for mutant sex factors with a greatly increased capacity for pilus production.     

R2R3型MYB转录因子HbMYB88结构和功能分析

R2R3-MYB转录因子参与植物生长发育、激素信号传导和逆境响应等生物过程。为了揭示橡胶树MYB家族成员结构与功能,从橡胶树热研73397（RY73397）叶片中克隆HbMYB88的cDNA全长。该基因长为1 848 bp,含1 440 bp的ORF,编码479个氨基酸。HbMYB88蛋白序列包含2个SANT保守结构域,存在HTH三级结构,与拟南芥AtMYB88、AtMYB124具有高度相似性。AtMYB88、AtMYB124与干旱等逆境相关且未划分亚族。qRT-PCR分析发现HbMYB88在橡胶树茎和花中表达量高,在根、叶片、树皮和胶乳中表达量极低。热研73397组培苗叶片喷施过氧化氢（H₂O₂）和脱落酸（ABA）等处理后,HbMYB88表达量受诱导显著上调表达。表明HbMYB88与橡胶树抗旱等逆境反应有关,为深入研究其结构和功能打下基础。