Antipain lethality to Escherichia coli: dependence upon cyclic adenosine 3'',5''-monophosphate and its receptor protein.

Antipain kills Escherichia coli K-12 cells in an exponential manner beginning 1 h after its addition. Mutant strains, delta cya and crp, which are unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) and the cAMP receptor protein, respectively, are not affected. Addition of cAMP (5 mM) to antipain-treated mutant strains causes killing of delta cya cells, but not crp cells. Thus the lethal effect of antipain is dependent upon cAMP and its receptor protein.     

Cyclic AMP and its receptor protein are required for expression of transfer genes of conjugative plasmid F in Escherichia coli

A number of cya and crp mutants of Escherichia coli HfrH were analyzed for several Tra functions of the F plasmid. The mutants were observed to be deficient in conjugal donor ability, absorption of phages MS2 and Q beta and surface exclusion. These defects were suppressed in cya mutants grown with cAMP supplementation. A cAMP concentration of 3 X 10(-4) M produced maximal suppression of donor ability defect in a cya strain. cAMP did not suppress the Tra- phenotype of crp mutants. Latent periods of MS2 were shorter in cya and crp bacteria. Phage T7 development appeared similar in wild type, cya, and crp cells. It is concluded that tra genes of F plasmid are expressed only to a small extent in cya and crp mutants and that cAMP and its receptor protein are required for the normal expression of tra genes.     

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.     

猪霍乱沙门氏菌C500株△crp△asd缺失株平衡致死载体系统的构建及鉴定

猪霍乱沙门氏菌C500株是用化学方法致弱、用于预防仔猪副伤寒的弱毒疫苗株,虽具有较好的免疫原性,但仍有一定的残余毒力。为了研制更加安全并保持C500株良好免疫原性的弱毒株,及将C500开发为适于粘膜免疫的疫苗活载体,本文构建了猪霍乱沙门氏菌C500株△crp△asd双缺失株平衡致死载体系统。首先构建含缺失320bp的crp(cAMP受体蛋白)基因与蔗糖敏感基因(sacB)的重组自杀性质粒,与C500接合转移,两步法筛选无抗性的△crp缺失株,用PCR证实基因组crp基因的缺失突变。用同样方法在crp缺失株基础上构建asd(天冬氨酸β-半乳糖脱氢酶)基因缺失株。该缺失株生长必需外源DAP(二氨基庚二酸)。进一步鉴定△crp缺失株的表型、生长特性、毒力等,结果表明△crp△asd缺失株构建成功。△crp△asd缺失株可以用来作为宿主载体平衡致死系统来高效表达外源基因,为深入研究以C500株为载体的口服多价疫苗奠定了基础。     

表达大肠杆菌LT-B/ST融合抗原的减毒鼠伤寒沙门氏菌株的构建

编码LT-B/ST融合抗原的基因插入pYA248载体中,构建了重组质粒pXZL66。该重组质粒转入无毒鼠伤寒沙门氏菌SR-11,ΔCya,Δcrp,Δasd菌株X4072。此无抗药性的杂合菌株X4072(pXZL66)表达的LT-B/ST融合抗原具有LT和ST抗原性而没有生物毒性,可望成为预防ETEC腹泻和相应的沙门氏菌病双价口服活疫苗候选株。     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

Generation of deletions in the 3'-flanking sequences of the Escherichia coli crp gene that induce cyclic AMP suppressor functions

The crp structural gene and its 3'-flanking sequences were subcloned into M13mp8, and in vitro deletions were constructed in both the 5' and 3' ends of the gene by using Bal 31 nuclease. Deletions ranged in size from 24 to 250 base pairs at the 5' end of crp. Sixteen deletions generated at the 3' end of the gene ranged in size from 133 to 675 base pairs. The majority of deletions extended into the crp structural gene. Another class of deletions, i.e., delta crp-4, delta crp-17, and delta crp-2, had endpoints extending in the 3'-flanking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Escherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the delta crp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed beta-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The delta crp-4 mutation also caused strain NCR440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the delta crp-4 mutation comigrated with the wild-type protein on electrophoretic gels. The delta crp-4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.     

Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli

We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.     

Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.     

Sites of allosteric shift in the structure of the cyclic AMP receptor protein

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.     

大肠杆菌棉子糖操纵子α—半乳糖苷酶表达的调节控制

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of CRP-mediated cya suppression in Escherichia coli.

Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene. NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control. In vivo beta-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP. The CRP isolated from NCR30 differed from the protein isolated from wild-type E. coli in many respects. The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP. Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation. The protein conferred a degree of cAMP independence on the in vitro synthesis of beta-galactosidase. In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo beta-galactosidase synthesis in NCR30. The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.