Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.     

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

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.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Escherichia coli K-12 ferrous iron uptake mutants are impaired in their ability to colonize the mouse intestine

Abstract The streptomycin-treated mouse colonization model was used to investigate the role of the Fe²⁺ uptake system (Feo) of Escherichia coli K12 in the colonization of the mouse intestine. Mutants impaired in the uptake of Fe²⁺ ions were shown to be deficient also in their colonization ability. Both enterochelin-producing and enterochelin-nonproducing Escherichia coli feo mutants were unable to colonize the mouse intestine. These results demonstrated that Fe(II) is an essential source of iron for E. coli grown in the intestine.     

Asymmetric crossing over in the spontaneous formation of large deletions in the tonB-trp region of the Escherichia coli K-12 chromosome.

The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA ⁺ and recA ⁻ cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79 × 10⁻⁹ and 1.09 × 10⁻⁹ for recA ⁺ and recA ⁻ cells, respectively. We analyzed 12 deletions from recA ⁺ and 10 from recA ⁻ cells by cloning and direct sequencing. The deletions ranged in size from 5612 bp to 15142 bp for recA ⁺ and from 5428 bp to 13289 for recA ⁻ cells. Three deletions from recA ⁺ cells and five deletions from recA ⁻ cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the repeat in the mutant sequence. Seven deletions from recA ⁺ cells and three deletions from recA ⁻ cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini in the wild-type are characterized by short (2–4 bp) repeats. From these results, a model is presented for the generation of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp. Received: 14 September 1998 / Accepted: 22 December 1998     

Strain and temperature-dependent variation in the amount of BtuB polypeptide in the Escherichia coli K-12 outer membrane

Abstract The outer membrane protein BtuB of Escherichia coli K-12 is the receptor for vitamin B₁₂; it is normally present in approx. 200 copies per cell. We describe here the conditions by which BtuB was readily observed in electropherograms of outer membrane preparations. These conditions are as follows. (1) Incorporation of 8 M urea in sodium dodecyl sulfate-polyacrylamide gel systems improved detection of the polypeptide. (2) In most E. coli K-12 strains examined, BtuB was most abundant when cells were grown at 27°C. This thermoregulation of BtuB was independent of envZ and envY , two regulatory genes for outer membrane proteins.     

Characterization of adhesion zones in E. coli cells

After plasmolysis of Escherichia coli cells, the adhesion zones were characterized using the cytochemical PTA and SP procedures which stain peptidoglycan and lipopolysaccharides (LPS) respectively. A PTA-stained layer was detected at the adhesion sites. This layer was visualized irrespective of the electron microscopy procedure used. Also, using SP staining an outer membrane in which LPS molecules were asymmetrically distributed, was observed.     

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.     

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12

Abstract Five different c -type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a funtional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c -type cytochrome was detected under any growth condition tested.     

大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.