AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Glyl0Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.     

Analysis of the flanking regions from different haemolysin determinants of Escherichia coli

Summary The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity pf the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phly _L) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A+T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A+T) but even the structural hly genes show a considerably higher A+T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A+T) suggesting that the hly determinant may not have originated in E. coli.Dedicated to Prof. F. Lingens on the occasion of his 60th anniversary     

An auto-inducible Escherichia coli lysis system controlled by magnesium

The Escherichia coli (E. coli) prokaryotic expression system is widely used in the field of biology. The currently adopted processes for inducing cell wall rupture, in order to release the target protein, are complex and cumbersome. We developed an auto-inducible E. coli lysis system that is regulated by exogenous magnesium ion (Mg²⁺) concentration. This system is composed of a strictly Mg²⁺-regulated promoter Pmgt from the mgtB gene of Salmonella typhimurium, and the lysis genes from λ bacteriophage. Both the wild type and Sam7-mutant lysis genes were inducibly expressed in E. coli under Mg²⁺-depleted conditions. The former caused a rapid lysis, while the latter induced very mild lysis of the host strains. However, rapid lysis was observed when the latter was resuspended in Tris–EDTA buffer. Finally, the inducible lysis cassette containing wild type lysis gene was introduced into an expression plasmid expressing GFP gene and efficient lysis of the host E. coli strain and subsequent release of the target protein was achieved in Mg²⁺-depleted conditions. Collectively, the current study indicates that this novel inducible lysis system could have attractive applications in the field of protein expression and provides new insights for the development of bacterium-based vaccines.     

Immunological detection of cloned antigenic genes of Vibrio cholerae in Escherichia coli

Antibodies have been used as probe to detect cloned genes coding for toxin and surface antigens of Vibrio cholerae E1 Tor strain KB207. Eco RI-digested chromosomal DNA of KB207 was cloned in plasmid pBR325 and transformed in Escherichia coli HB 101(λcI857). Transformants were grown at 32° C on plates containing antibodies. Lysogen was induced at 42 °C to release expressed antigens. Antigen-antibody reaction produced a halo around positive clones.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12

Summary Mutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded -lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins. The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E. coli linkage map. A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups. Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others.     

In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3′-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3′-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm. Received: 7 April 1997 / Accepted: 28 May 1997     

Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Oligomerization of membrane-bound Bcl-2 is involved in its pore formation induced by tBid

Both pro-apoptotic Bax and anti-apoptotic Bcl-2 are structurally homologous to the pore-forming domain of bacterial toxins. Bax proteins oligomerize in the mitochondrial outer membranes forming pores that release cytochrome c from the mitochondrial intermembrane space. Bcl-2 proteins also form pores that, however, are much smaller than the Bax pore. It is unknown whether Bcl-2 forms monomeric or oligomeric pores. Here, we characterized the Bcl-2 pore formation in liposomes using biophysical and biochemical techniques. The results show that the Bcl-2 pore enlarges as the concentration of Bcl-2 increases, suggesting that the pore is formed by Bcl-2 oligomers. As expected from oligomerization-mediated pore-formation, the small pores are formed earlier than the large ones. Bcl-2 oligomers form pores faster than the monomer, indicating that the oligomerization constitutes an intermediate step of the pore formation. A Bcl-2 mutant with higher affinity for oligomerization forms pores faster than wild type Bcl-2. Bcl-2 oligomers were detected in the liposomal membranes under conditions that Bcl-2 forms pores, and the extent of oligomerization was positively correlated with the pore-forming activity. Therefore, Bcl-2 oligomerizes in membranes forming pores, but the extent of oligomerization and the size of the resulting pores are much smaller than that of Bax, supporting the model that Bcl-2 is a defective Bax.     

Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12

Summary Strains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium. Wild-type genes defined by these mutations have been cloned from E. coli genomic bank made with plasmid pBR328. Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes. Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.     

Recombinant Wheat Antifungal PR4 Proteins Expressed in Escherichia coli

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

ALT: A new factor involved in the synthesis of RNA by Escherichia coli

Summary We have defined a new gene, alt, which affects RNA synthesis in Escherichia coli. Mutants for alt arise among revertants of strains lacking the CRP-cAMP system necessary for full expression of catabolite-sensitive operons. Studies on a temperature-sensitive alt mutant indicate that the alt gene product is necessary for the synthesis of an important class of messenger RNA molecules.     

Introduction of a Micrococcus plasmid in Escherichia coli

A 6-MDa plasmid (pMQV10), carrying cholesterol hydroxylase and streptomycin-resistance genes, from a gram-positive strain of Micrococcus sps., (RJ6) has been successfully transformed in gram-negative Escherichia coli K12 C600. pMQV10 is maintained stably and expresses its drug resistance in the new host.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary In spite of the generally well-coordinated synthesis of RNA polymerase core enzyme subunits (, and ) in Escherichia coli, a situation was found during the growth transition from exponential to stationary phase in which this coordination was broken (the order of differential repression being ; Kawakami et al. (1979)). The present study indicates that, during a certain period of the growth transition, twice as much subunit is synthesized as subunit and the overproduced subunit accumulates as the assembly intermediate ₂ complex, which is rapidly and preferentially degraded.Two independent factors, i.e., carbon source down-shift and oxygen depletion, were examined separately for their influence on the coordinated regulation of the synthesis of RNA polymerase subunits. The depletion of glucose added as a sole carbon source was accompanied by repression of the synthesis of all core enzyme subunits, while under the same conditions the differential rate of subunit synthesis increased. In contrast, the sudden ending of the oxygen supply resulted in specific repression of the synthesis of only and subunits but not of and subunits. The latter result may be explained by the autogenous repression of the rpoBC genes by a temporal increase in the amount of unused cytoplasmic RNA polymerase.Paper XI in this series is Kawakami and Ishihama (1980)