Saccharomyces cerevisiae killer expression mutant kex2 has altered secretory proteins and glycoproteins.

The extracellular proteins and glycoproteins of a yeast mutant $\text{kex}\text{2–15}$ defective in killer toxin expression were separated by one and two dimensional polyacyylamide gel electrophoresis. Many mutant extracellular proteins and glycoproteins show both altered electrophoretic mobility and isoelectric points when compared with the parent strain. Altered proteins and glycoproteins from $\text{kex}\text{2–15}$ were identified with their parental counterparts by peptide mapping. The observed alterations co-segregated with the $\text{kex}\text{2}$ nuclear mutation in genetic crosses.     

Ribosomal mutation in Escherichia coli affecting membrane stability

Summary Mutations in ribosomal protein L6 cause (i) loss of viability of cells at 0° C, which can be prevented by the presence of sodium chloride or 20% sucrose in the medium, (ii) influx of compounds at low temperature that normally cannot penetrate, and (iii) a defective assembly and maturation of 30S and 50S subunits at low temperature. It is proposed that abnormal interaction of immature subunits (or mutant 70S ribosomes) with the cytoplasmic membrane is responsible for triggering breakdown of membrane stability during cold shock.     

Homology between Drosophila melanogaster and Escherichia coli ribosomal proteins

Summary Antibodies raised against D. melanogaster ribosomal proteins were used to examine possible structural relationships between eukaryotic and prokaryotic ribosomal proteins. The antisera were raised against either groups of ribosomal proteins or purified individual ribosomal proteins from D. melanogaster. The specificity of each antiserum was confirmed and the identity of the homologous E. coli ribosomal protein was determined by immunochemical methods. Immuno-overlay assays indicated that the antiserum against the D. melanogaster small subunit protein S14 (anti-S14) was highly specific for protein S14. In addition, anti-S14 showed a cross-reaction with total E. coli ribosomal proteins in Ouchterlony double immunodiffusion assays and with only E. coli protein S6 in immuno-overlay assays. From these and other experiments with adsorption of anti-S14 with individual purified proteins, the E. coli protein homologous to the D. melanogaster protein S14 was established as protein S6.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary The effect on translational fidelity of a particular mutation in the gene coding for protein S5 (rpxE) has been investigated. This mutation has the opposite effect of a restrictive strA mutation; in vivo, it relieves the restriction imposed by strA on the suppression of T4 nonsense mutants and results in hypersensitivity to streptomycin; in vitro, the presence of the altered S5 protein in 30S ribosomes results in increased intrinsic misreading. It is concluded that this mutation, ramC₃₁₉, acts as a ribosomal ambiguity mutation similar to certain mutations of protein S4 (ramA).     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

The stoichiometry of the ribosomal proteins of Escherichia coli

Summary A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

利用重组大肠杆菌合成几丁寡糖的研究

提取根瘤菌Mesorhizobium.loti基因组,克隆编码N-乙酰氨基葡萄糖转移酶nodC基因,插入质粒pUC19的lac启动子的下游,构建并筛选出能够合成几丁寡糖的重组大肠杆菌DCL-3。利用优化的MMYNG培养基,重组大肠杆菌DCL-3在10L发酵罐中培养26h后,培养液菌体浓度测定OD560=10.8,几丁寡糖得率达到526mg/L。收集重组细菌的细胞并煮沸破碎,利用活性炭的吸附和P4凝胶层析对几丁寡糖产物进行分离纯化。纯化产物的液质分析(LC-ESI-MS)结果表明主要寡糖产物为几丁四糖(m/z,831[M H] )和几丁五糖(m/z,1034[M H] )。     

Transfection of Escherichia coli by Mu DNA

Summary Infectivity of Mu DNA was demonstrated in Ca⁺⁺-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10^-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.     

Synthesis of Escherichia coli outer membrane OmpA protein in yeasts

Saccharomyces cerevisiae was transformed with the Escherichia coli ompA gene coding for an outer membrane protein. Yeast transformants containing the pYTLJ101 plasmid, consisting of the ompA gene cloned in pSC101 and the HindIII-3 fragment of 2-μm DNA, express the foreign membrane protein. The protein synthesized in yeast has an M_r value very similar if not identical to that of the mature E. coli protein. The expressed protein is present in yeast mitochondrial and plasma membrane fractions. The yeast cell can tolerate about 250 molecules of the foreign membrane protein per cell, although the transformants show altered growth kinetics.     

Genetic studies of the ribosomal proteins in Escherichia coli X

Summary Episomes of E. coli, which cover argG but not the str region, were transferred to Serratia marcescens. Ribosomal proteins from these hybrid strains were analyzed with phospho-cellulose or carboxymethyl-cellulose column chromatography. Two E. coli ribosomal proteins, L21 and S15, could be detected in the ribosome from the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli

Summary In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles.It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25°, the methyl group is stable when the culture is shifted to 37°C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25°C. On the other hand, the methyl groups of L11 appear to be stable at either 25°C or 37°C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.     

Release of outer membrane fragments from normally growing Escherichia coli

A complex containing lipopolysaccharides, phospholipids and proteins is released into the culture medium by Escherichia coli during normal growth. It can be separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation. Electron microscopy revealed that this material is released as vesicles and membrane fragments. To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes. The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane. Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.     

探索大肠杆菌细胞膜合成过程中脂肪酸的掺入模式

【目的】探索大肠杆菌生长分裂过程中,脂肪酸作为底物在细胞膜合成过程中的掺入模式。【方法】本研究解析了以乙酰CoA为底物,合成中间产物长链脂酰-ACP,随后合成磷脂酰乙醇胺(phosphatidylethanolamine,PE)的途径,并将合成途径中的10个关键酶与绿色荧光蛋白(enhanced green fluorescent protein,EGFP)或红色荧光蛋白(monmer Cherry,mCherry)进行融合,在大肠杆菌内表达这些融合蛋白,用激光共聚焦荧光显微镜成像的方式来获得这些融合蛋白的定位信息。【结果】宽场荧光显微镜成像结果显示,磷脂酰乙醇胺合成途径中的10个酶在不同表达水平下出现不同的定位模式。在大肠杆菌中高水平表达融合蛋白EGFP-FabA、EGFP-FabB、EGFP-FabI、EGFP-FabG、EGFP-PlsB和EGFP-PssA时,细胞两极和中部有大量蛋白聚集的现象。EGFP-FabD、EGFP-FabF、EGFP-CdsA、EGFP-PSD在不同表达水平下,均匀分散在细胞质或细胞膜上。缩时影像(Time-lapse)结果显示,合成途径中的一个关键蛋白EGFP-Pls B在细胞分裂前随着细胞膜的内陷聚集到细胞隔膜,随着细胞分裂,母细胞的隔膜成为新细胞的两极。【结论】本研究通过获取磷脂酰乙醇胺合成相关蛋白酶在大肠杆菌中的定位结果,推测脂肪酸分子是在细胞分裂隔膜和两极掺入,被催化合成PE后被运送到细胞膜其他位置。     

Genes affecting the major outer membrane proteins of Escherichia coli K-12: Mutations at nmpA and nmpB

Summary Strains of Escherichia coli K-12 carrying mutations at either of two distinct loci (nmpA and nmpB) produce a new outer membrane pore protein which is not present in wild type cells. Mutations at either of these loci result in sensitivity to phage TC45, which can use this new protein as its receptor, and the new protein (the NmpAB protein) appears to be identical in both NmpA and NmpB mutants. In order to determine whether both of these loci contain structural genes for the NmpAB protein, strains carrying secondary mutations at either of these loci which produced altered proteins were sought by selecting for resistance to phage TC45. Mutants which produced proteins with altered electrophoretic mobility and altered peptide maps were isolated from strains carrying both nmpA and nmpB mutations, and these secondary mutations mapped at the same sites as the original mutations leading to production of the NmpAB protein. This suggests that both loci contain structural genes. Strains mutant at nmpB which can no longer produce the protein can mutate at the nmpA locus to produce the new protein, indicating that both genes can exist in the same cell. Since the altered proteins of mutant strains could be distinguished from one another, we attempted to construct strains in which both nmpA and nmpB were expressed. In all cases only the protein produced by the nmpB mutation was produced, indicating some form of cooperative regulation of the two genes.