Colicin M is inactivated during import by its immunity protein

Colicin M (Cma) displays a unique activity that interferes with murein and O-antigen biosynthesis through inhibition of lipid-carrier regeneration. Immunity is conferred by a specific immunity protein (Cmi) that inhibits the action of colicin M in the periplasm. The subcellular location of Cmi was determined by constructing hybrid proteins between Cmi and the TEM--lactamase (BlaM), which confers resistance to ampicillin only when it is translocated across the cytoplasmic membrane with the aid of Cmi. The smallest Cmi'-BlaM hybrid that conferred resistance to 50 g/ml ampicillin contained 19 amino acid residues of Cmi; cells expressing Cmi'-BlaM with only five N-terminal Cmi residues were ampicillin sensitive. These results support a model in which the hydrophobic sequence of Cmi comprising residues 3–23 serves to translocate residues 24–117 of Cmi into the periplasm and anchors Cmi to the cytoplasmic membrane. Residues 8–23 are integrated in the cytoplasmic membrane and are not involved in Cma recognition. This model was further tested by replacing residues 1–23 of Cmi by the hydrophobic amino acid sequence 1–42 of the penicillin binding protein 3 (PBP3). In vivo, PBP3'-'Cmi was as active as Cmi, demonstrating that translocation and anchoring of Cmi is not sequence-specific. Substitution of the 23 N-terminal residues of Cmi by the cleavable signal peptide of BlaM resulted in an active BlaM'-'Cmi hybrid protein. The immunity conferred by BlaM'-'Cmi was high, but not as high as that associated with Cmi and PBP3'-'Cmi, demonstrating that soluble Cmi lacking its membrane anchor is still active, but immobilization in the cytoplasmic membrane, the target site of Cma, increases its efficiency. Cmi1-23 remained in the cytoplasm and conferred no immunity. We propose that the immunity protein inactivates colicin M in the periplasm before Cma can reach its target in the cytoplasmic membrane.     

Functional domains of colicin M

The structure of colicin M of Escherichia coli was studied with regard to its organization into functional domains. A proteolytic fragment with an M_r of 24,000 was isolated which comprised the carboxyterminal portion of the protein. It adsorbed to the outer membrane receptor protein and inhibited killing of cells by colicin M and by phage T5 that uses the same receptor. The fragment killed cells when the outer membrane was rendered permeable to macromolecules for a short time by the osmotic shock procedure. It is concluded that the fragment contains the receptor binding site and the active center but is lacking the sequence required for transport into cells.The carboxy-terminal amino acid sequence-Lys-Arg of the fragment was identical to that obtained from colicin M. Release of lysine and arginine led to inactivation of colicin M. The sequence of the first 39 amino acids of the amino terminal end of colicin M was determined.Abbreviation EGTA ethylene glyco-bis (-aminoethyl ether)-N-N-tetraacetate     

Domains of colicin M involved in uptake and activity

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the Tong, ExbB and ExbD proteins. These colicins contain a sequence, called the Tong box, which has been implicated in transport via Tong. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with Tong may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This Colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193–197 and 223–252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the Tong box at the N-terminal end of colicin M must be involved in colicin uptake via Tong across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

Requirement of the SecB chaperone for export of a non-secretory polypeptide in Escherichia coli

Summary The SecB protein of Escherichia coli is a cytosolic component of the export machinery which can prevent some precursors from prematurely folding into export-incompatible conformations by binding to the newly synthesised polypeptide. The feature(s) of target proteins recognised by SecB, however, are unclear and have been a matter of controversy. Also, it has not been asked if binding of SecB is specific for secretory proteins. We demonstrate here that a non-secretory polypeptide, a fragment of a tail fiber protein of phage T4, fused to the signal peptide of the outer membrane protein OmpA has a very strong SecB requirement for export and that the signal peptide itself cannot, at least not alone, be responsible for this action of SecB. The data reported, together with those of the literature, suggest that SecB recognizes the polypeptide backbone of the target protein.     

Experimental confirmation of a key role for non-optimal codons in protein export

Non-optimal codons are defined by low usage and low abundance of corresponding tRNA, and have an established role in translational pausing to allow the correct folding of proteins. Our previous work reported a striking abundance of non-optimal codons in the signal sequences of secretory proteins exported via the sec-dependent pathway in Escherichia coli. In the current study the signal sequence of maltose-binding protein (MBP) was altered so that non-optimal codons were substituted with the most optimal codon from their synonymous codon family. The expression of MBP from the optimized allele (malE-opt) was significantly less than wild-type malE. Expression of MBP from malE-opt was partially restored in a range of cytoplasmic and periplasmic protease deficient strains, confirming that reduced expression of MBP in malE-opt was due to its preferential degradation by cytoplasmic and periplasmic proteases. These data confirm a novel role for non-optimal codon usage in secretion by slowing the rate of translation across the N-terminal signal sequence to facilitate proper folding of the secreted protein.     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.     

Secretion and export of IGF-1 in Escherichia coli strain JM101

Summary The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.     

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.     

Plasmid distribution in Escherichia coli urinary isolates with special reference to aerobactin and colicin production

Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified cases of urinary tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed an association of aerobactin and colicin production with the distribution of plasmid bands having a median value of 2.33 and 1.72 (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For colicin producers, the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains.     

E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。     

影响微生物互作的重要基因研究进展：以大肠杆菌为例

微生物在自然界中广泛存在,微生物间的相互作用对群落结构和功能有重要影响。目前已经对微生物相互作用的机制给予了很大的关注,通过高通量测序技术和统计学分析方法的结合可以定位获得影响菌株互作的重要基因。为了深入研究微生物相互作用的遗传机制,本文以大肠杆菌(Escherichia coli)为例,综述了与大肠杆菌运动性、耐药性、营养物质吸收和代谢调节相关的基因在互作条件下发挥的作用,并从这几个方面分别阐述了大肠杆菌互作遗传机制。总之,这些基因在大肠杆菌与其他微生物互作中发挥重要作用,同时增强了对细菌互作机制的理解,为今后研究更复杂的微生物群落互作遗传机制奠定了理论基础。     

不同致泻性大肠杆菌感染调控细胞信号通路的研究进展

腹泻性大肠杆菌是在全世界引起人类和动物疾病的主要病原之一,也给社会经济带来巨大损失。根据致病机理的不同,可将腹泻性大肠杆菌分为6种:肠致病性大肠杆菌、肠出血性大肠杆菌、肠凝集性大肠杆菌、产肠毒素大肠杆菌、扩散黏附性大肠杆菌和肠侵袭性大肠杆菌。不同致病型大肠杆菌侵入宿主的方式及引起的炎症反应有所不同。文章综合分析了致病机制不同的大肠杆菌在调控宿主细胞信号通路方式上的不同,从炎症级联反应方面阐述了不同致病类型大肠杆菌的感染特征,并探讨了炎症信号通路与病原感染、预防和治疗的关系,以期为腹泻性大肠杆菌致病机制及治疗方案的研究提供帮助。     

和厚朴酚对大肠埃希氏菌生物被膜形成的抑制机制

【目的】研究不同浓度的和厚朴酚(honokiol)抑制大肠埃希菌(Escherichia coli)的供试菌株10389生物被膜(biofilm,BF)形成的作用机制。【方法】用氯化三苯基四氮唑比色法(TTC)和四唑盐减低法(XTT)测定honokiol抑制E.coli10389生物被膜形成的药物最低抑菌浓度(MIC)和最低杀菌浓度(MBC)及其抑制作用与时间的关系;通过qRT-PCR法检测不同浓度的honokiol对E. coli 10389生物被膜形成基因和群体感应系统相关基因表达量的影响;通过生物发光法和qRT-PCR法检测亚-MIC honokiol对E. coli 10389呋喃糖基硼酸二酯(AI-2)及其调控的与生物被膜形成相关的下游基因表达量的影响。【结果】Honokiol能抑制E.coli10389生物被膜的形成,但不同浓度的honokiol抑制E. coli 10389 BF形成的作用机制不同。其中,与对照组相比,MIC的honokiol能使E. coli 10389 BF形成相关基因编码毒素(hha)和细菌酸性调节因子(ari R) mRNA的表达量显著提高,抗毒素...     

Parasitism of Iron-siderophore Receptors of Escherichia Coli by the Siderophore-peptide Microcin E492m and its Unmodified Counterpart

Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniae RYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJ gene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-l-serine (DHBS) linked to the Ser84-carboxylate via a β-d-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor affinity.