Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein

Summary The ccd operon of plasmid F encodes two genes, ccdA and ccdB, which contribute to the high stability of the plasmid by post-segregational killing of plasmid-free bacteria. The CcdB protein is lethal to bacteria and the CcdA protein is an antagonist of this lethal action. A 520 by fragment containing the terminal part of the ccdA gene and the entire ccdB gene of plasmid F was cloned downstream of the tac promoter. Although the CcdB protein was expressed from this fragment, no killing of host bacteria was observed. We found that the absence of killing was due to the presence of a small polypeptide, CcdA41, composed of the 41 C-terminal residues of the CcdA protein. This polypeptide has retained the ability to regulate negatively the lethal activity of the CcdB protein.     

Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein, in this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.     

Improved seamless mutagenesis by recombineering using ccdB for counterselection

Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.     

Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.     

Directed evolution of copy number of a broad host range plasmid for metabolic engineering

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.     

Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H(+)-ATPase C,G and d subunits,and their evaluation in autosomal recessive distal renal tubular acidosis

Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide. The ability of inteins to promote protein splicing even when inserted into a foreign context has facilitated the study of the modulation of protein splicing. In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis. It involves the activation of the cytotoxic CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing is blocked. The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZ alpha-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by protein splicing. Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion protein. To induce large amounts of CcdB fusion proteins, the fusion protein is expressed from a high-copy-number plasmid. Such a screening system detects even low levels of protein splicing and we have used it to show that protein splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction. In order to search for protein splicing inhibitors, which may attenuate protein splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when splicing of the expressed CcdB fusion protein is only moderately suppressed. We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of protein splicing.     

Intricate interactions within the ccd plasmid addiction system.

The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA*CcdB*DNA complex with a CcdA:CcdB ratio of one. This (CcdA(2)CcdB(2))(n) complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.     

Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.     

Structural and functional analysis of the kid toxin protein from E. coli plasmid R1

We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.     

Parallel assembly for multiple site-directed mutagenesis of plasmids

A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants-larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction-ligation reaction.     

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.     

An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae

Insertional mutagenesis is a technique often used to inactivate genes in Streptococcus pneumoniae. Using conventional vectors, a 5' segment of the targeted gene remains under the control of the gene's authentic promoter following gene disruption. Thus, the expression of a functional peptide and the misinterpretation of results in consequence cannot be excluded. To circumvent this problem, we have developed a plasmid for insertional mutagenesis based on the tmRNA-tagging system of S. pneumoniae which ensures that any protein expressed after gene disruption is degraded. Insertional mutagenesis using this vector results in the targeted gene being tagged with a tmRNA-derived sequence coding for a proteolysis tag. Here we show that the translation product of a gene tagged by this method is not detectable by Western blotting, suggesting that the protein was degraded. This modified vector allows total inactivation of genes with a reliability that cannot be achieved by conventional vectors for insertional mutagenesis. This approach can be applied to other bacterial species.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.