Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Influence of the culture conditions on extracellular lyase activity related to K5 polysaccharide

The K5 capsular polysaccharide antigen of some Escherichia coli strains is the non-sulphated precursor in heparin biosynthesis. It is composed by two components, 16000 and 1500 Da, whose ratio depends on the activity of the extracellular form of a lyase synthesized by the same K5 producer strain. The lyase activity on the K5 chain size was greatly influenced by the medium composition employed for the lyase production. The control of lyase activity results in defined ratios of the two components in the K5 polysaccharide that is suitable for the semisynthetic production of heparin-like molecules.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.     

K5裂解酶在大肠杆菌中的表达、纯化及酶学性质分析

K5多糖裂解酶(Elma)能够裂解半合成肝素的底物-K5多糖,裂解产物是半合成法生产低分子量肝素的底物。利用PCR方法扩增elma,构建表达载体pET-28a-Elma,将构建好的质粒转化至大肠杆菌BL21中,以0.2 mmol/L的IPTG在16℃诱导5 h实现了高效表达,SDS-PAGE分析表明Elma表达量可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法和G-75分子筛层析纯化目的蛋白,其纯度大于95%。通过PAGE多糖电泳发现裂解前后的K5多糖分子量有明显的减小。根据Elma裂解产物产生双键从而在232 nm处有吸光度的变化来测Elma的酶活。其最适反应温度为37℃,反应的最适pH值为7.0。底物特异性分析发现Elma除K5多糖外对肝素和透明质酸也有降解作用。     

Glutathione-gated K+ channels of Escherichia coli carry out K+ efflux controlled by the redox state of the cell

The kinetics of K⁺ efflux across the membranes of i) wild-type Escherichia coli poisoned by the thiol reagent N-ethylmaleimide, ii) K⁺ retention mutants and iii) glutathione-deficient mutants, have revealed a common K⁺ leaky phenotype; it is characterized by a very high rate of K⁺ efflux. The results suggest that the products of kefB and kefC genes could encode two K⁺ channels, both gated by glutathione. The possible function of these K⁺ channels seems to be a K⁺ exit controlled by the redox state of the cell; indeed, it can be inferred from the effects of several oxidants and reductants that turning on and off of the K⁺ efflux mediated by the channels can be correlated with the redox state of glutathione.     

Expression and immunogenicity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> K99 fimbriae subunit antigen in soybean

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T₀ events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T₀ lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T₁ and T₂ generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4⁺ T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.     

A naturally occurring large chromosomal inversion in Escherichia coli K12

Summary Strain 1485IN and its derivatives were found to have a large inversion extending to about 35% of the chromosome. Because of this, the question arose as to whether 1485IN had arisen from an Escherichia coli strain other than K12. However, 1485IN had a flagellar antigen and a restriction-modification system indistinguishable from those of W3110, a major line of K12, and had retained an amber suppressor and sensitivity that are characteristics of W1485 from which this strain seems to have arisen. Strain 1485IN had acquired proline auxotrophy, but showed the same growth rate as W1485 in nutrient broth at 37°C. Interrupted matings with Hfr strains of 1485IN revealed a gene arrangement of nalA-gal-trp-his-lac-proA-thrleu-ilv, in which gal, trp, and his were on the inverted segment. The termini of the inversion were inferred to be situated between tsx (9.5 min) and purE (12 min) and between his (44 min) and cdd (46.5 min).     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

Recombinant streptokinase production by fed-batch cultivation of Escherichia coli

Streptokinase (SK) is a specific effective medicine for thrombolytic therapy of acute myocardial infarction. This study established a process for the pilot production of recombinant streptokinase (r-SK). Engineering bacteria were fermented in a 20-l fermentor to produce r-SK. After simple renaturation and purification, 12.9 g of r-SK with 97.8% of purity and about 10⁵ IU mg⁻¹ of specific activity was obtained, the yield of protein and the recovery of activity were 44.9% and 51%, respectively. Finally, the r-SK was made into about 700 doses of injections for clinical applications.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Assessing the toxic effect of 2,4,6-trinitrotoluene on cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> K12 by flow cytofluorometry

The magnitude of transmembrane potential Δψ in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of Δψ in cells. In the presence of 200 mg/l of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of Δψ over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18–20 mg/l). The mean value of Δψ in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger fraction of the subpopulation with a low value of Δψ. Upon elimination of TNT, the distribution of Δψ in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

荧光标记噬菌体快速检测K1荚膜大肠杆菌方法的建立及应用

【背景】大肠杆菌(Escherichia coli,E.coli)是引发新生儿脑膜炎和禽类脑膜炎最常见的革兰氏阴性菌,其中含K1荚膜大肠杆菌是重要的病原菌。目前,K1荚膜大肠杆菌的检测方法存在一些弊端。【目的】利用PNJ1809-36噬菌体的宿主特异性建立快速检测K1荚膜大肠杆菌的方法。【方法】用荧光染料SYBR Gold标记PNJ1809-36噬菌体,侵染33株受试菌,在荧光显微镜下观察,测定该方法的特异性;倍比稀释宿主菌DE058,用荧光标记噬菌体侵染,测定该方法的灵敏度;用荧光标记噬菌体检测8份模拟粪样,测定该方法的临床应用效果;测定4℃避光保存4个月的荧光标记噬菌体的效价和检测效果。【结果】33株受试菌中的9株K1荚膜大肠杆菌有8株可见环状荧光,1株未能检出;20株非K1荚膜大肠杆菌以及4株非大肠杆菌属细菌均不能观察到荧光,检测灵敏度达100CFU/mL。8份模拟粪样的检测结果显示,3份含有K1荚膜大肠杆菌的粪样均可见环状荧光,5份不含K1荚膜大肠杆菌的粪样均无荧光。荧光标记噬菌体4℃避光保存4个月后效价无明显下降,检测效果无明显变化,表明该荧光标记噬菌体在4℃避光条件下较稳定...     

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Summary Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharification was 86.3 ± 1.5 g/L. The pretreated WS was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. The recombinant Escherichia coli strain FBR5 produced 41.1 ± 1.1 g ethanol/L from non-abated WS hydrolyzate (total sugars, 86.6 ± 0.3 g/L) in 168 h at pH 7.0 and 35 °C. The bacterium produced 41.8 ± 0.0 g ethanol/L in 120 h from the bioabated WS by SHF. It produced 41.6 ± 0.7 g ethanol/L in 120 h from bioabated WS by fed-batch SSF. This is the first report of the production of above 4% ethanol from a lignocellulosic hydrolyzate by the recombinant bacterium.     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

毒力岛基因ibeT有助于大肠杆菌抵抗人脑微血管内皮细胞溶酶体的降解

大肠杆菌是导致新生儿细菌性脑膜炎最常见的革兰氏阴性致病菌．为探讨毒力岛基因ibeT在大肠杆菌K1株致病过程中的作用,构建了ibeT基因缺失的大肠杆菌K1株,细菌在细胞内存活试验结果显示,ibeT基因缺失抑制了大肠杆菌K1株在人脑微血管内皮细胞中的生长．利用激光共聚焦扫描显微镜观察到,在细菌侵袭进入人脑微血管内皮细胞后,与野生型相比,ibeT基因缺失突变株较多地滞留在溶酶体内;透射电镜结果进一步显示,ibeT基因缺失使大肠杆菌K1株逃逸ECV(含有大肠杆菌的囊泡)的能力发生了下降,继而使其在细胞浆内的复制减少．利用体外模拟的弱酸性环境,检测大肠杆菌菌体胞内的缓冲容量,发现ibeT基因缺失突变株菌体胞内的缓冲能力较野生型低．这些结果提示,在大肠杆菌K1株侵袭进入人脑微血管内皮细胞后,ibeT基因有利于大肠杆菌降解ECV膜,避免与溶酶体融合,进而促使大肠杆菌逃逸进入细胞浆并进行复制．     

口服微生态制剂对孕妇肠道和阴道大肠杆菌K1和B族链球菌定殖的干预

【目的】本研究主要从临床探讨口服益生菌对孕妇微生态环境(阴道和肠道)中Escherichia coli K1和B族链球菌(GBS)定殖率的影响以预防新生儿血流播散性细菌性脑膜炎。【方法】收集2011年至2017年期间在广东省范围门诊就诊的2539例妊娠健康孕妇的阴道、直肠分泌物。选择符合要求的32孕周孕妇47例随机分成两组,其中益生菌组22例,对照组25例,用荧光定量PCR检测不同组孕妇体内服药前、后的微生态环境中大肠埃希菌的变化。然后选择50例GBS筛查阳性的35孕周孕妇,随机分成益生菌组和对照组,荧光定量PCR检测不同分组孕妇体内微生态环境中GBS定量变化。同时使用荧光PCR筛查对临床收集的2539例孕晚期(35周)孕妇生殖道、直肠分泌物标本进行GBS检测并计算携带率。【结果】研究前对不同组别孕妇的基本资料进行差异分析,结果显示两组在年龄差别、经产妇比例和受教育水平3个方面比较均无差异(P0.05),证明不同组间孕妇具有可比性。然后观察上述不同组别的孕妇服用益生菌体内微生态状况,用荧光定量PCR方法进行检测,结果显示用益生菌组在服药前后,阴道和直肠分泌物中大肠杆菌数量显著下降(F=32.866,P0.001),孕妇服用益生菌后E. coli K1的数量显著低于服用益生菌前(P0.05),且益生菌组与对照组相比大肠杆菌数量显著下降(P0.05,F=41.546,P0.001);同时检查服益生菌组孕妇体内GBS的变化,荧光定量PCR检测结果显示服用益生菌前后GBS的定殖数量有降低趋势,且与对照组相比有下降趋势。最后我们用荧光定量PCR筛查孕晚期妇女B族链球菌带菌状况,其GBS携带率为8.07%。【结论】本研究结果提示本文筛查的广东省孕晚期孕妇的B族链球菌平均携带率为8.07%,GBS可能是新生儿发生细菌性脑膜炎的危险因素之一;口服益生菌疗法可能通过抑制E. coli K1在孕妇肠道和生殖道的定殖以预防血流播散性新生儿细菌性脑膜炎。