Methylation strongly enhances DNA bending in the replication origin region of the Escherichia coli chromosome

Summary Two-dimensional gel electrophoresis, at high and low temperatures, and gel mobilities of circularly permuted DNA segments showed a large bending locus about 50 bp downstream from the right border of the 245 by oriC box, a minimal essential region of autonomous replication on the Escherichia coli chromosome. Bending was strongly enhanced by Dam methylation. In DNA from a Dam^– strain, the mobility anomaly arising from altered conformation was much reduced, but was raised to the original level by methylation in vivo or in vitro. Enhancement of the mobility anomaly was also observed by hybrid formation of the Dam^– strand with the Dam⁺ strand. Near the bending center, GATC, the target of Dam methylase, occurs seven times arranged essentially on the same face of the helix with 10.5 by per turn. We concluded that small bends at each Dam site added up to the large bending detectable by gel electrophoresis.     

Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system

Summary Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth indam ⁺ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyldirected mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage X174 containing either 0, 1, or 2 GATC sequences, in wild type,dam, andmut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterialmutH ⁺ dam^– strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type andmutL andmutS bacteria whereas the effect is not significant inmutU bacteria, suggesting an interaction of the, helicase II with the MutH protein.However, indam ⁺ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield inmutH ^– bacteria reveals that methylated GATC sequences are advantageous to the phage. These results suggest that the methyl-directed mismatch repair system, and in particular its MutH protein, may have participated in severe counterselection of GATC sequences from enterobacteriophages, presumably, by DNA cleavage or by interfering with DNA replication or packaging when GATC sequences are undermethylated. Coevolution of the Dam and MutH proteins could then account for the loss of GATC sequences from DNA of bacteriophages growing indam ⁺ hosts.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H₆TLHS1, was purified using a Ni²⁺ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H₆TLHS1 was produced to follow the fate of H₆TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H₆TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H₆TLHS1 to the E. coli cells without H₆TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H₆TLHS1 survived about three thousand times more than the bacterial cells without H₆TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.     

Characterisation of cisplatin coordination sites in cellular <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl₂(NH₃)₂] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b⁺ and y⁺ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.     

Characterization of Cold-Shock Protein A of Antarctic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. AA8321

Abstact Polar organisms should have mechanisms to survive the extremely cold environment. Four genes encoding cold-shock proteins, which are small, cold-induced bacterial proteins, have been cloned from the Antarctic bacterium Streptomyces sp. AA8321. Since the specific functions of any polar bacterial or Streptomyces cold-shock proteins have not yet been determined, we examined the role of cold-shock protein A from Streptomyces sp. AA8321 (CspA_St). Gel filtration chromatography showed that purified CspA_St exists as a homodimer under physiological conditions, and gel shift assays showed that it binds to single-stranded, but not double-stranded, DNA. Overexpression of CspA_St in Escherichia coli severely impaired the ability of the host cells to form colonies, and the cells developed an elongated morphology. Incorporation of a deoxynucleoside analogue, 5-bromo-2′-deoxyuridine, into newly synthesized DNA was also drastically diminished in CspA_St-overexpressing cells. These results suggest that CspA_St play a role in inhibition of DNA replication during cold-adaptation.     

优化的绿色荧光蛋白在超嗜热嗜酸古菌冰岛硫化叶菌中的表达与应用

【目的】开发可用于在极端嗜热嗜酸模式泉古菌冰岛硫化叶菌(Sulfolobus islandicus)中进行高效表达的eCGP123(enhanced consensus green protein variant 123)荧光蛋白,并用作S.islandicus的细胞内蛋白定位工具。【方法】绿色荧光蛋白突变体eCGP123具有极高的热稳定性、耐酸性和可逆的荧光特性等。本研究主要对eCGP123的基因根据S.islandicus密码子偏好性进行优化与合成,在大肠杆菌(Escherichia coli)中表达并研究其蛋白性质;通过在eCGP123的C末端分别融合具有不同细胞内定位的蛋白(包括E.coli来源的Fts Z和S.islandicus来源的Ups E、PCNA1和SiRe_1200等),构建eCGP123及其融合蛋白的表达菌株,用激光共聚焦显微镜分析eCGP123及其融合蛋白在E.coli和S.islandicus活细胞中的亚细胞定位。【结果】我们确认了在E.coli中表达并纯化密码子优化后的e CGP123具有与野生型绿色荧光蛋白相同的吸光值和较高的热稳定性。细胞学分析显示细胞分裂相关蛋白FtsZ和Si Re_1200分别主要定位于E.coli和S.islandicus分裂细胞的中间;鞭毛组分蛋白Ups E呈点状均匀分布,可能定位于细胞膜上;DNA复制滑动夹亚基PCNA1呈区域性点状分布,暗示了DNA复制区域的位置。蛋白的亚细胞定位与预期结果基本吻合。【结论】绿色荧光蛋白e CGP123可以作为报告蛋白,应用于S.islandicus细胞的蛋白定位分析中,可作为该模式菌株中功能基因研究的重要工具,但需要进一步优化条件。     

Enhanced tolerance against freezing stress in<Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing an algal cyclophilin gene

Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions. To study the functions of the cDNAGjCyp-1 isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed inEscherichia coli. Most of the gene product expressed inE. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration ofl(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 inE. coli were examined. The results indicate that the induction of Cyp, at 0.2%l(+)-arabinose for 2 h at 25°C, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase. An expressed fusion protein, H₆GjCyp-1, maintained the stability ofE. coli proteins up to-75°C. In a functional bioassay of the recombinant H₆GjCyp-1, the viability ofE. coli cells overexpressing H₆GjCyp-1 was compared to that of cells not expressing H₆GjCyp-1 at −75°C. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in theE. coli cells overexpressing H₆GjCyp-1. The results of the GjCyp-1 bioassays, as well asin vitro studies, strongly suggest that the algal Cyp confers freeze tolerance toE. coli.     

Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences.

Summary We previously demonstrated that the E. coli protein, H-NS (or Hla), encoded by the gene hns (or osmZ or bglY preferentially recognizes curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.     

Characterization of a dUTPase from the Hyperthermophilic Archaeon <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80°C and pH 8.0, and it was highly thermostable with a half-life (t _1/2) of 170 min at 95°C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl₂, KCl, (NH₄)₂SO₄, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli

Cytochrome b₅ (b₅) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b₅ on CYP-catalyzed reactions, but also that of the apo-cytochrome b₅ (apo-b₅). Therefore, the apo-b₅ protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b₅ was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b₅ was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b₅ was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b₅ protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b₅ protein was obtained. Oxidized and reduced forms of the apo-b₅ reconstituted with heme exhibit the same absorbance spectra as native b₅. The prepared recombinant apo-b₅ reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b₅ is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b₅.     

Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

cDNA cloning,expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III

Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and -irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA

Summary A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M₁ progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M₂ progenies, obtained by self-fertilization of dwarf M₁ plants, were also dwarf, while those from tall M₁ plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m⁵C) content in comparison with DNA from untreated samples. A similar reduction in the m⁵C content was also observed in the M₁ and M₂ progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.