The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.     

Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.     

The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase

The maturation pathway of microcin B17 (MccB17), a ribosomally synthesized peptide antibiotic which inhibits DNA gyrase, has been characterized. Synthesis of MccB17 involves several steps beginning with the translation of the MccB17 structural gene, mcbA, to yield a 69 amino acid precursor, preMccB17. PreMccB17 is then modified and folded by the action of three gene products, McbBCD, to yield proMccB17. Mutations in mcbA were isolated that permit modifications of the resulting mutant peptides, but prevent folding, suggesting that modification and folding are sequential steps. ProMccB17 is subsequently converted to MccB17 by removal of the W-terminal 26-amino-acid leader by a chromosomally encoded protease. Removal of the leader resulted in aggregation of the peptide, suggesting that the leader may function to maintain peptide solubility during synthesis in the cell. Finally, polyclonal antibodies raised against MccB17 recognize both MccB17 and proMccB17, but do not recognize preMccB17. This demonstrates the dramatic structural changes that result from the modifications and has been used to distinguish intermediates in the steps of maturation.     

The action of the bacterial toxin, microcin B17, on DNA gyrase

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.     

The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.     

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.     

Novel method of expression and purification of hirudin based on pBAD TOPO, pTYB12 vectors and gene synthesis

To express recombinant hirudins in Escherichia coli cells, a fragment of chemically synthesized DNA was used, containing codons for the individual amino acids preferred by the host cells. Gene synthesis was based on the design of two DNA fragments, so-called mega primers H1 and H2 with a complementary fragment, and their incubation with Taq polymerase. The gene obtained in this fashion was multiplied using the PCR, and then expressed in E. coli cells with the use of TOPO vectors pBAD and pTYB12. Using this method, hirudins were obtained in the amount of 17 mg/l E. coli strain, with the activity of 17 antithrombin units (ATU)/mg protein. The method can be considered as an easy and inexpensive route to small protein synthesis.     

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase–DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial–bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.     

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.     

Characterizing the DNA contacts and cooperative binding of F plasmid TraM to its cognate sites at oriT

TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.     

The export of the DNA replication inhibitor Microcin B17 provides immunity for the host cell.

Microcin B17 (MccB17) is a peptide antibiotic which inhibits DNA replication in Enterobacteriaceae. Microcin-producing strains are immune to the action of the microcin. Physical and genetic studies showed that immunity is mediated by three genes: mcbE, mcbF and mcbG. We sequenced these genes and identified polypeptide products for mcbF and mcbG. By studying the contribution of each gene to the expression of immunity we found that immunity is determined by two different mechanisms. One of these, encoded by mcbE and mcbF, is also involved in the production of extracellular MccB17. To reconcile these observations we propose that McbE and McbF serve as a 'pump' for the export of active MccB17 from the cytoplasm. This model is supported by the predicted properties of the McbE and McbF proteins, which are thought to be, respectively, an integral membrane protein and an ATP-binding protein with homology to other transport proteins.     

Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.     

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.