Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.     

大肠杆菌周质蛋白提取工艺的改进

介绍一种简捷高效的大肠杆菌周质蛋白提取工艺,即用含一定浓度溶菌酶的细胞裂解缓冲液一步提取周质蛋白,与传统的高渗和低渗两步提取法相比,不仅操作简单快捷,并且显著的提高了大肠杆菌周质蛋白的提取率.     

大肠杆菌抗铜蛋白高表达的条件

含有抗铜质粒pRJ1004的大肠杆菌(Escherichia coli)可以在含有20mmol/L的LB培养基中生长,菌落呈深棕色,其原因是由于一种“修饰铜”沉淀的结果。以同位素64~Cu所做的试验表明,抗性菌株比敏感菌株积累铜少,其抗性机制是减少吸收。质粒pRJ1004含一个抗铜基因组:pcoABCDRSE,,其中pcoABCDE为结构基因,pcoRS为调节基因。pcoABCD的基因产物和丁香假单胞菌(Pseudomonas syringae pv.tomato)的copABCD基因产物很相似,有很高的氨基酸序列同源性。而后者的菌落在含铜平板上则呈深蓝色,且细胞中可积累比敏感菌株更多的铜,可达细胞干重的0.3％。copABCD基因产物在细胞中的分布及功能都已研究得比较清楚,它们都是铜结合蛋白。     

表达载体pHsh对大肠杆菌热休克系统中负调控机制的影响

pHsh是一种由σ32识别和启动外源基因表达的新型高效的大肠杆菌表达载体。正常E.coli细胞在热激诱导条件下,σ32的浓度在5 min内到达高峰,随后被3个负调控蛋白Dnak、DnaJ、GrpE结合导致失活或降解,整个热休克反应持续约12min。在携带有外源基因的高拷贝pHsh 的E.coli细胞中,外源基因却能持续高效表达4～10 h,这一现象表明了此时细胞中的σ32比没有携带质粒的细胞内σ32的浓度要高。σ32浓度的增高有可能是由于3个负调控蛋白Dnak、DnaJ、GrpE在细胞内的含量比正常情况下降低的结果。为了验证这一推测,从E.coli中克隆了Dnak、DnaJ、GrpE的编码基因,表达并初步纯化了其重组蛋白以作分子标记,采用双向电泳技术,分析携带质粒（pHsh+）和不携带质粒的E.coli(pHsh-)细胞在热休克后胞内蛋白质组的差异。该项实验通过与检索到的标准的E.coli蛋白质组图谱进行比较鉴别出的两个蛋白Dnak、GrpE,并通过对比目标点的大小和深浅发现pHsh+中的Dnak均少于pHsh─中的目标蛋白,所得结果与上述假设一致。     

Gram-positive cell wall structure of the A3γ type in heliobacteria

Abstract The periplasmic enzyme β-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals. At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred. Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH. At intermediate concentrations of tetradecyl betainate high yields of β-lactamase were obtained with no detectable contribution of the cytoplasmic marker β-galactosidase. The highest yields of β-lactmase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate.     

Purification and processing of cellulose-binding domain-alkaline phosphatase fusion proteins

Fusion of the leader peptide and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from Cellulomonas fimi, with of without linker sequences, to the N-terminus of alkaline phosphatase (PhoA) from Escherichia coli leads to the accumulation of significant amounts of the CBD-PhoA fusion proteins in the supernatants of E. coli cultures. The fusion proteins can be purified from the supernatants by affinity chromatography on cellulose. The fusion protein can be desorbed from the cellulose with water or guanidine-HCl. If the sequence IEGR in present between the CBD and PhoA, the CBD can be cleaved from the PhoA with factor Xa. The efficiency of hydrolysis by factor Xa is strongly in fluenced by the amino acids on either side of the IEGR sequence. The CBD released by factor Xa is removed by adsorption to cellulose. A nonspecific proteases from C. fimi, which hydrolyzes native CenA between the CBD and the catalytic domain, may be useful for removing the CBD from some fusion proteins. (c) 1994 John Wiley & Sons, Inc.     

大肠杆菌周质蛋白提取工艺的研究

研究了精氨酸缓冲液在不同浓度、pH值及提取时间下的周质蛋白提取率,并以溶菌酶法、渗透压休克法作为对比。结果表明浓度0.4 mol/L,pH值8.0,提取时间为45 min时,周质目的蛋白达到0.89 mg/g湿菌,相比其他方法,周质蛋白提取率分别提高93%、187%。实验得到一种高效、方便的大肠杆菌周质蛋白提取工艺,为周质表达的重组蛋白大规模生产奠定了基础。     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.     

Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium

AIMS: The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance. METHODS AND RESULTS: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1). We also show that all E. coli cells are inherently capable of growth on deuteriated media. CONCLUSIONS: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains. SIGNIFICANCE AND IMPACT OF STUDY: An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.     

Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k_on 2.82×10⁷ M^–1 s^–1 and k_off 4.49×10³ s^–1. The dissociation constant of the primary O₂ compound was 160 M (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K_m <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.This paper is dedicated to Professor Dr Hans G. Schlegel, on the occasion of his 80th birthday.     

Outer membrane targeting of secretin PulD protein relies on disordered domain recognition by a dedicated chaperone

Interaction of bacterial outer membrane secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for secretin function in type II secretion. Although other secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between secretins and their chaperones.     

Impairment of cell division in tolA mutants of Escherichia coli at low and high medium osmolarities.

Mutations in the tolA gene of Escherichia coli cause the cell to become sensitive to detergents and to some antibiotics, to release periplasmic enzymes and to be resistant to group A colicins; tolA mutations also lead to mucoid phenotype. TolA is a three-domain protein anchored in the inner membrane by its N-terminal domain. The second domain is proposed to span the periplasmic space and to interact with trimeric porins of the outer membrane. TolA proteins are considered to be located in the adhesion zones between inner and outer membranes. Our observations by confocal and electron microscopy have revealed that tolA mutants show modified morphology and produce DNA-free cells. Increasing or decreasing medium osmolarity amplifies these defects; mutants become essentially unable to locate the division site properly so that cells of highly unequal lengths are produced. Moreover, septation is impaired with asymmetric constrictions and oblique septa. These results suggest that TolA could play a role in positioning the division sites via the organisation of either the outer membrane or the possible adhesion zones.     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置:胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和用途 。     

Loss of O-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli

Escherichia coli mutants lacking multiple penicillin-binding proteins (PBPs) produce aberrantly shaped cells. However, most of these experiments have been performed in E. coli K12 strains, which do not attach a complete O-antigen to their outer membrane lipopolysaccharide. We constructed mutants in different genetic backgrounds and found that the frequency of morphological deformities was higher in strains lacking the O-antigen. Also, complementing O-negative mutants with a heterologous O-antigen from Klebsiella returned a substantial fraction of misshapen cells to a normal morphology. Thus, the O-antigen contributes to cell shape in E. coli, perhaps by reducing the number of ectopic poles, which may be the proximal cause of shape abnormalities.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.