Calculation of entropy change accompanying growth of Escherichia coli K-12 on succinic acid

The ΔS of one unit carbon formula weight of Escherichia coli K-12 cells, when grown on succinic acid, was calculated to be ?80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K-12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.     

On the enthalpy of formation of Escherichia coli K-12 cells

An examination is made of five methods for obtaining values of the enthalpy of formation of a unit mass of living Escherichia coli K-12 cells. The values obtained by these methods ranged from -88.95 kJ to -99.55 kJ, the gross average being 96.01 kJ, per unit carbon formula weight equivalent of living, hydrated cells. Although theoretically the growth of this organism in a microcalorimeter should provide the best value, the value obtained by this method (-88.95 kJ per UCFW equivalent) is not in close agreement with those of the other four methods, the values from which form a cluster averaging -97.8 +/- 1.0 kJ (-23.4 +/- 0.2 kcal)/UCFW equivalent. Calculations using this value indicate that the enthalpy change accompanying anabolism (as this is represented) is zero, or very nearly so, and that the heat of growth is that from catabolism alone.     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

大肠杆菌σ70启动子的识别

应用多样性增量结合二次判别分析（Increment of Diversity with Quadratic Discriminant analysis,IDQD）方法,对大肠杆菌σ^70启动子进行识别。使用受试者操作特性（receiver operating characteristic,ROC）曲线和精度召回率曲线（Precision Recall Curves,PRC）进行性能评估。10-fold交叉检验给出,在正负集之比为1：1时,ROC曲线下面积和PRC曲线下面积均为95％。结果表明,IDQD算法有能力应用于原核启动子的识别。识别精度高于现有算法。     

Calculation of the heat of growth of Escherichia coli K-12 on succinic acid

Using data from the literature, a method is adopted for determining the empirical composition and the unit carbon formula for dried Escherichia coli K-12 cells by summing the quantities of C, H, O, N, P, and S in each of the major classes of macromolecular substances comprising the cellular biomass. With these data and the molar growth yield of cells on succinic acid, equations are written representing the anabolism and catabolism of E. coli K-12 on this quantity of substrate. The enthalpy change accompanying catabolism can be calculated directly using standard enthalpies of formation because there is no term representing cellular substance. The enthalpy change accompanying anabolism is calculated to be very small or zero using microcalorimetric and other data from which the enthalpy of formation of a unit quantity of living cellular substance can be obtained. This indicates that the net enthalpy change accompanying the growth process (anabolism plus catabolism) is the same as that calculated for catabolism alone, in agreement with the same conclusion by several investigators using direct microcalorimetry. The method described here of determining the unit carbon formula and the quantity of ash remaining after cellular combustion is compared to that conventionally used in which cellular P and S is considered either to be negligible or to be a part of the ash. It is concluded that equations representing anabolism and the growth process can be written more accurately using the presently described method, leading to more accurate thermodynamic calculations.     

Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

A 718-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 12.7-28.0 min Region on the Linkage Map

The 718,122 base pair (bp) sequence of the Escherichia coliK-12 genome corresponding to the region from 12.7 to 28.0 minuteson the genetic map is described. This region contains at least682 potential open reading frames, of which 278 (41%) have beenpreviously identified, 147 (22%) were homologous to other knowngenes, 138 (20%) are identical or similar to the hypotheticalgenes registered in databases, and the remaining 119 (17%) didnot show a significant similarity to any other gene. In thisregion, we assigned a cluster of cit genes encoding multienzymecitrate lyase, two clusters of fimbrial genes and a set of lysogenicphage genes encoding integrase, excisionase and repressor inthe e14 genetic element. In addition, a new valine tRNA gene,designated valZ, and a family of long directly repeated sequences,LDR-A, -B and -C, were found.     

A 460-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 40.1-50.0 min Region on the Linkage Map

The 465,813 base pair sequence corresponding to the 40.1–50.0min region on the genetic map of Escherichia coli K-12 (W3110)was determined. Analysis of the sequence revealed that thisregion contained at least 466 potential open reading frames,of which 187 (40%) were previously reported, 105 (23%) werehomologous to other known genes, 103 (22%) were identical orsimilar to hypothetical genes registered in databases, and theremaining 71 (15%) did not show a significant similarity toany other gene. At the 45.2–46.0 min region, we founda very large cluster of about 30 genes, whose functions areinvolved in the biosynthesis of polysaccharides as the componentsof outer membranes. In addition, we identified anew asn-tRNAgene, designated asnW, between the asnT and asnU genes and anew lysogenic phage attachment site as the cis-element.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Induction of cadmium tolerance in Escherichia coli K-12

Abstract Cadmium ions are bacteriocidal, resulting in exponential killing that starts immediately after exposure. We have shown that pretreatment with sublethal concentrations of cadmium induces tolerance. Protection against cadmium killing can also be obtained by preincubation at elevated temperatures, known to induce the heat-shock response. However, in contrast to pretreatment at elevated temperatures, exposure to sublethal cadmium concentrations does not induce thermotolerance.     

Bidirectional frequency-dependent effect of extremely low-frequency electromagnetic field on E. coli K-12

In the present work, the frequency-dependent effects of extremely low-frequency electromagnetic field (ELF EMF) on Escherichia coli K-12 growth have been studied. The frequency-dependent effects of ELF EMF have shown that it can either stimulate or inhibit the growth of microbes. However, the mechanism by which the ELF EMF affects the bacterial cells is not clear yet. It was suggested that the aqua medium can serve as a target through which the biological effect of ELF EMF on microbes may be realized. To check this hypothesis, the frequency-dependent effects (2, 4, 6, 8, 10 Hz, B = 0.4 mT, 30 min) of ELF EMF on the bacterial growth were studied in both cases where the microbes were in the culture media during the exposure and where culture media was preliminarily exposed to the ELF EMF before the addition of bacteria. For investigating the cell proliferation, the radioactive [³H]-thymidine assay was carried out. It has been shown that EMF at 4 Hz exposure has pronounced stimulation while at 8 Hz it has inhibited cell proliferation.     

A 570-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 28.0-40.1 min Region on the Linkage Map

The 569,750 base pair sequence corresponding to the 28.0–40.1min region on the genetic map of Escherichia coli K-12 (W3110)was determined. This region includes the replication terminusregion and contained at least 549 potential open reading frames.Among them, 160 (29%) were previously reported, 174 (32%) werehomologous to other known genes, 102 (18%) were identical orsimilar to hypothetical genes registered in databases, and theremaining 113 (21%) did not show a significant similarity toany other gene. Of interest was the finding of a large numberof genes and gene clusters in andnear the replication terminationregion which had been thought to be genetically silent. Thoseincludeda cluster of genes for fatty acid ß-oxidation,the third copy of the pot (spermidine/putrescine transport system)gene cluster, the second dpp (dipeptide transport system) operon,the second dsm (anaerobic dimethyl sulfoxide reductase) operon,a cluster of fim (fimbrial) genes anda DNA helicase-like genewith a high molecular weight. In addition, we found the dnaC-and dnaT-like genes in the cryptic prophage, Rac, anda numberof genes originated probably from plasmids.     

Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon.

Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides. Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes. Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively.     

大肠杆菌酸性磷酸酶基因克隆表达及应用

将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.     

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and , -dipyridyl but not from the siderophores ferrichrome and enterochelin.     

Isolation and preliminary characterization of β-lactamase excretory mutants of Escherichia coli K-12

Abstract Escherichia coli exc mutants able to release the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium have been isolated using a new in situ plate assay.
A preliminary characterization of the exc mutants was carried out: the presence of exc mutations was associated with a specific or pleiotropic pattern of excretion of periplasmic enzymes, an increased sensitivity to different growth inhibitors (EDTA, chloramphenicol, cholic acid) and a poor growth on various carbon sources.
After quantitative analysis, three groups of exc mutants were identified on the basis of their temperature-dependent or -independent pattern of growth and β-lactamase synthesis and excretion.     

Four critical aspartic acid residues potentially involved in the catalytic mechanism of Escherichia coli K-12 WaaR

Escherichia coli K-12 WaaR is a non-processive alpha-1,2 glucosyltransferase, involved in the synthesis of the R-core of lipopolysaccharide. WaaR possesses the four conserved structural regions I, II, III and IV, each presumably involved in the mechanistic function in catalysis. Regions I and III contain the pair of strictly conserved Asp residues. Asp-129, 131 (region I) and 215, 217 (region III) of WaaR were individually converted to Asn by the site-directed mutagenesis of the waaR gene. All mutated enzymes were inactive, supporting the model for an alpha-glycosyl transfer reaction where the pair of strictly conserved aspartic acid residues in regions I and III play a critical role in the catalytic function.