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.     

Identification of Messenger Ribonucleic Acids and Proteins Synthesized by the Bacteriocinogenic Factor Clo DF13 in Purified Minicells of Escherichia coli

It has previously been shown that the cloacinogenic factor Clo DF13 (Clo DF13) segregates into minicells of strain Escherichia coli P678-54 that harbors Clo DF13 and that this Clo DF13 factor is the only deoxyribonucleic acid (DNA) present in these otherwise chromosomeless minicells. The study reported here shows that minicells prepared from P678-54(Clo DF13) are able to incorporate radioactive precursors into ribonucleic acid (RNA) and protein. The RNA synthesized in these purified minicells is Clo DF13 specific, as shown by RNA-DNA hybridization experiments. The results indicate that all the de novo synthesized gene products in Clo DF13 minicells are Clo DF13 specific. Polyacrylamide gel electrophoretic patterns show that in these minicells at least three polypeptides (molecular weight about 70,000, 20,000, and 11,000) and one major species of messenger RNA (mRNA) (S value about 21.3) are synthesized. To investigate the factor in its induced state, we isolated a Clo DF13 mutant with an enhanced level of cloacin production. Minicells harboring this Clo DF13 mutant produce five additional polypeptides (molecular weight about 58,000, 44,000, 28,000, 16,000, and 14,000). Three additional mRNA species (S value about 19.5, 14, and 12) could be distinguished. The total molecular weight of the eight polypeptides corresponds to 85% of the total coding capacity of the mRNAs (303,000). The total molecular weight of the four mRNAs is 2.55 x 10(6), which covers 85% of the Clo DF13 DNA (molecular weight 6 x 10(6)).     

Molecular Studies on Entry Exclusion in Escherichia coli Minicells

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).     

Unusual Membranous Structures in Minicells and Minicell-Producing Strains of Escherichia coli

Minicell-producing strains of Escherichia coli K-12 seem to produce extra membranous material yielding internal cross membranes, “piggy-back” minicell forms, and unusual vesicles.     

Presence of Nuclear Bodies in Some Minicells of Escherichia coli

Electron microscopy of serial sections of Escherichia coli K-12 (P678-54) revealed that some "minicells" contain nuclear bodies.     

Membrane Association of Conjugally Transferred Deoxyribonucleic Acid in Escherichia coli Minicells

The conjugally acquired deoxyribonucleic acid (DNA) of small, anucleate cells ("minicells") of a mutant strain of Escherichia coli K-12 was found to be predominantly associated with the bacterial membrane. Evidence from X-irradiation studies in vivo shows that there is no decrease in DNA-membrane association under conditions which reduce the DNA to one-sixth its original size and suggests the possibility of multiple DNA-membrane association sites. Preliminary enzymatic studies indicate the involvement of protein, DNA, and lipids in the membrane association of the DNA.     

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.     

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.     

Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in E. coli.     

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.     

Enzymatic Reduction of Mercurous Ions in Escherichia coli Bearing R Factor

Mercurous ion (Hg(+))-dependent reduced nicotinamide adenine dinucleotide phosphate oxidation was demonstrated in an extract from cells of Escherichia coli W2252 that bear R factor.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

Isolation of Membrane-Associated Folded Chromosomes from Escherichia coli: Effect of Protein Synthesis Inhibition

The sedimentation properties of membrane-associated folded chromosomes prepared from Escherichia coli TAU-bar at 0 to 4 C were studied. Utilizing a modification of the procedure of Stonington and Pettijohn (1971), quantitative yields of membrane-associated folded chromosomes may be obtained. Folded chromosomes remained associated with the cell envelope during their replication and after completion of residual synthesis in the absence of required amino acids, as demonstrated by sedimentation velocities and the presence of high levels of cosedimenting protein. Membrane-associated folded chromosomes isolated from amino acid-starved cells sedimented more rapidly than membrane-associated folded chromosomes isolated from exponentially growing cells.     

Physical and Functional Characteristics of R-Factor Deoxyribonucleic Acid Segregated into Escherichia coli Minicells

Both fi(+) and fi(-) type R factors function as intact replicative units when segregated into Escherichia coli minicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R(+) minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R(+) minicells in contrast to R(-) minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

Proteins induced by anaerobiosis in Escherichia coli

The contribution of protein induction and repression to the adaptation of cells to changes in oxygen supply is only poorly understood. We assessed this contribution by measuring the levels of 170 individual polypeptides produced by Escherichia coli K-12 in cells growing aerobically or anaerobically with and without nitrate. Eighteen reached their highest levels during anaerobic growth. These 18 polypeptides include at least 4 glycolytic enzymes and pyruvate formate-lyase (beta-subunit). Most of these proteins were found at significant levels during aerobic growth and appeared to undergo metabolic regulation by stimuli other than anaerobiosis. Anaerobic induction ratios ranged from 1.8- to 11-fold, and nitrate antagonized the anaerobic induction of all of the proteins except one. The time course of synthesis of the proteins after shifts in oxygen supply revealed at least three distinct temporal patterns. These results are discussed in light of known physiological alterations associated with changes in oxygen availability.     

Methylation of Ribosomal Proteins in Escherichia coli

Escherichia coli was grown in a medium containing [1-(14)C]methionine and [methyl-(3)H]methionine, and the (3)H/(14)C ratio was determined for each of the ribosomal proteins derived from the 70S ribosome. Evidence indicates that six proteins from the 50S subunit were methylated: L7, L9, L11, L12, L18, and L33. Methylation of several other 50S proteins (such as L1, L3, L5, etc.) may also occur. The methylated amino acids in protein L11 have been characterized further and found to be predominately epsilon-trimethyllysine. A small amount of a compound tentatively identified as N(G), N'(G)-dimethylarginine was also detected.     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.