Single-crossover Recombination Research Articles

Development and application of a rapid gene manipulating toolbox for Pseudomonas aeruginosa

Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.

Targeted Mutagenesis of Mycobacterium Strains by Homologous Recombination.

Targeted mutagenesis by homologous recombination (TMHR) is an efficient allelic exchange mutagenesis for bacterial genome engineering in synthetic biology. Unlike other allelic exchange methods, TMHR does not require a heterologous recombinase to insert or excise a selectable marker from the genome. In contrast, positive and negative selection is achieved solely by suicide vector-encoded functional and host cell proteins. Here we describe a concise protocol to knock out and knock in a 3-ketosteroid-1,2-dehydrogenase gene (kstd) in Mycobacterium neoaurum HGMS2 using TMHR approach. The homology arms flanking the kstd gene are amplified by PCR in vitro and then subcloned into a common homologous recombination vector. The vector is then electroporated into the HGMS2 competent cells. The replacement of the kstd gene by homologous recombination produces antibiotic-resistant single-crossover recombination via the first allelic exchange. Double-crossover markerless mutants are directly separated using sucrose-mediated counterselection. These two steps can generate seamless mutations down to a single DNA base pair. The whole process takes less than 2weeks.

Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii

The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a “dual integrase cassette exchange” (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.

Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9

BackgroundTargeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.ResultsWe generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17–0.45%) that was used to knock in an approximately 5-kb long DNA fragment.ConclusionWe developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.

Application of methylation in improving plasmid transformation into Helicobacter pylori

Helicobacter pylori is an important gastrointestinal pathogen. Its strains possess different levels of powerful restriction modification systems, which are significant barriers to genetic tools used for studying the role of functional genes in its pathogenesis. Methylating vectors in vitro was reported as an alternative to overcome this barrier in several bacteria. In this study we used two H. pylori-E. coli shuttle plasmids and several single/double-crossover homologous recombination gene-targeting plasmids, to test the role of methylation in H. pylori transformation. According to our results, transformants could be obtained only after shuttle plasmids were methylated before transformation. It is helpful in gene complementation and over-expression although at a low frequency. The frequency of gene-targeting transformation was also increased after methylation, especially for the single-crossover recombination plasmids, the transformants of which could only be obtained after methylation. For the double-crossover recombination targeting plasmids, the initial yield of transformants was 0.3–0.8 × 102 CFUs per microgram plasmid DNA. With the help of methylation, the yield was increased to 0.4–1.3 × 102 CFUs per microgram plasmid DNA. These results suggest that in vitro methylation can improve H. pylori transformation by different plasmids, which will benefit the pathogenic mechanism research.

A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production.

We herein adapted a markerless gene replacement method by combining a temperature-sensitive plasmid pKSV7 with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the poly-γ-glutamic acid (γ-PGA)-producing strain Bacillus amyloliquefaciens LL3. Deletion of the upp gene conferred LL3 5-fluorouracil (5-FU) resistance. Sensitivity to 5-FU was restored when LL3 Δupp was transformed with pKSV7-based deletion plasmid which carries a functional allele of the upp gene of Bacillus subtilis 168. These observations allowed us to adapt a two-step plasmid integration and excision strategy to perform markerless deletion of genes of interest. Deletion plasmid harboring a mutant allele of the target gene was first integrated in the genome by culturing cells under nonpermissive conditions for pKSV7 replication. Single-crossover recombinants were then grown without antibiotics to aid the second recombinational event. 5-FU was used to select for double-crossover recombinants with plasmid evicted from the chromosome. The resulting recombinants either harbored the wild-type or mutated allele of the target gene and could be identified by PCR and DNA sequencing. Using this method, we successively removed the amyA gene and a 47-kb fragment of the bae cluster from the genome of LL3, with higher efficiency compared with previous reports. We also investigated the effects of a transcriptional regulator, RocR, on γ-PGA production and cell growth. Specific γ-PGA production of the rocR mutant was increased by 1.9-fold, which represents a new way to improve γ-PGA production.

Characterization of a methyltransferase involved in herboxidiene biosynthesis

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.

MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE lpdA1 GENE IN S. meliloti

The alfalfa-Sinorhizobium symbiosis is one of the best studied for plant-microbe associations. The pyruvate dehydrogenase complex catalyzes the key metabolic step connecting the glycolysis to the Tricarboxylic acid (TCA) cycle. The PDH also participates in anaplerotic synthesis of acetyl-CoA in bacteroids, or when grown in the presence of dicarboxylic acids as the sole carbon sources. The complex consists of multiple copies of the three subunits, pyruvate dehydrogenase (EC 1.2.4.1), that acts as the E1 component of the complex, dihydrolipoamide transacetylase (EC 2.3.1.12), the E2 component, and dihydrolipoamide dehydrogenase (EC 1.8.1.4), that participates as the E3 subunit. The lpdA1 gene was mutated through the site-directed, single crossover recombination, using modified pVIK112 suicide plasmids. The lpdA1 mutant failed to grow on pyruvate, and was significantly delayed on other tested carbon sources. The activity of the pyruvate dehydrogenase complex was not detected in this mutant, and the regulation of the lpdA1 was not dependent on the presence of pyruvate. This mutant was Nod+, but appeared Fix-.

Single-crossover recombination and ancestral recombination trees

We consider the Wright-Fisher model for a population of [Formula: see text] individuals, each identified with a sequence of a finite number of sites, and single-crossover recombination between them. We trace back the ancestry of single individuals from the present population. In the [Formula: see text] limit without rescaling of parameters or time, this ancestral process is described by a random tree, whose branching events correspond to the splitting of the sequence due to recombination. With the help of a decomposition of the trees into subtrees, we calculate the probabilities of the topologies of the ancestral trees. At the same time, these probabilities lead to a semi-explicit solution of the deterministic single-crossover equation. The latter is a discrete-time dynamical system that emerges from the Wright-Fisher model via a law of large numbers and has been waiting for a solution for many decades.

Plasmid integration method: a new tool for analysis of the essentiality and function of genes in S. aureus

Staphylococcus aureus is a Gram-positive coccus and one of the major causes of community‐acquired and hospital-acquired infections. We established the convenient and reliable experimental system for analyzing the essentiality and function of genes, the plasmid integration (PI) method. This method is based on plasmid integration into the genome by single cross-over recombination using a temperature-sensitive shuttle vector, and it was validated using known essential genes, gyrA and mvaD, and non-essential genes, sigB and hla. Then we analyzed 116 S. aureus conserved hypothetical protein genes with the PI method, and identified 28 essential genes. Moreover, applying the PI method, we confirmed the functional redundancy between the S. aureus gene (SA0865) and its ortholog human gene, the NAD kinase gene. These results show that the PI method is a powerful tool for the identification of essential genes and functional analysis by evaluation of complementarity.

Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum

We have shown that transgenic Plasmodium falciparum parasites expressing the yeast DHODH (dihydroorotate dehydrogenase) are independent of the mtETC (mitochondrial electron transport chain), suggesting that they might not need the mitochondrial genome (mtDNA), since it only encodes three protein subunits belonging to the mtETC and fragmentary ribosomal RNA molecules. Disrupting the mitochondrial RNA polymerase (mtRNAP), which is critical for mtDNA replication and transcription, might then cause the generation of a ρ0 parasite line lacking mtDNA. We made multiple attempts to disrupt the mtRNAP gene by double crossover recombination methods in parasite lines expressing yDHODH either episomally or integrated in the genome, but were unable to produce the desired knockout. We verified that the mtRNAP gene was accessible to recombination by successfully integrating a triple HA tag at the 3′ end via single cross-over recombination. These studies suggest that mtRNAP is essential even in mtETC-independent P. falciparum parasites.

Centromere Plasmid: A New Genetic Tool for the Study of Plasmodium falciparum

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

BackgroundAcinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate A. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of A. baumannii.ResultsWe describe here a rapid and simple method of obtaining A. baumannii mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (omp33, oxyR, and soxR) by this method. In addition, we disrupted one of these genes (omp33) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining A. baumannii mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in A. baumannii.ConclusionsThis study provides a rapid and simple method of obtaining stable mutants of A. baumannii free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.

Single-crossover recombination in discrete time

Modelling the process of recombination leads to a large coupled nonlinear dynamical system. Here, we consider a particular case of recombination in discrete time, allowing only for single crossovers. While the analogous dynamics in continuous time admits a closed solution (Baake and Baake in Can J Math 55:3-41, 2003), this no longer works for discrete time. A more general model (i.e. without the restriction to single crossovers) has been studied before (Bennett in Ann Hum Genet 18:311-317, 1954; Dawson in Theor Popul Biol 58:1-20, 2000; Linear Algebra Appl 348:115-137, 2002) and was solved algorithmically by means of Haldane linearisation. Using the special formalism introduced by Baake and Baake (Can J Math 55:3-41, 2003), we obtain further insight into the single-crossover dynamics and the particular difficulties that arise in discrete time. We then transform the equations to a solvable system in a two-step procedure: linearisation followed by diagonalisation. Still, the coefficients of the second step must be determined in a recursive manner, but once this is done for a given system, they allow for an explicit solution valid for all times.

A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.

A decomposition theory for phylogenetic networks and incompatible characters.

Phylogenetic networks are models of evolution that go beyond trees, incorporating non-tree-like biological events such as recombination (or more generally reticulation), which occur either in a single species (meiotic recombination) or between species (reticulation due to lateral gene transfer and hybrid speciation). The central algorithmic problems are to reconstruct a plausible history of mutations and non-tree-like events, or to determine the minimum number of such events needed to derive a given set of binary sequences, allowing one mutation per site. Meiotic recombination, reticulation and recurrent mutation can cause conflict or incompatibility between pairs of sites (or characters) of the input. Previously, we used "conflict graphs" and "incompatibility graphs" to compute lower bounds on the minimum number of recombination nodes needed, and to efficiently solve constrained cases of the minimization problem. Those results exposed the structural and algorithmic importance of the non-trivial connected components of those two graphs. In this paper, we more fully develop the structural importance of non-trivial connected components of the incompatibility and conflict graphs, proving a general decomposition theorem (Gusfield and Bansal, 2005) for phylogenetic networks. The decomposition theorem depends only on the incompatibilities in the input sequences, and hence applies to many types of phylogenetic networks, and to any biological phenomena that causes pairwise incompatibilities. More generally, the proof of the decomposition theorem exposes a maximal embedded tree structure that exists in the network when the sequences cannot be derived on a perfect phylogenetic tree. This extends the theory of perfect phylogeny in a natural and important way. The proof is constructive and leads to a polynomial-time algorithm to find the unique underlying maximal tree structure. We next examine and fully solve the major open question from Gusfield and Bansal (2005): Is it true that for every input there must be a fully decomposed phylogenetic network that minimizes the number of recombination nodes used, over all phylogenetic networks for the input. We previously conjectured that the answer is yes. In this paper, we show that the answer in is no, both for the case that only single-crossover recombination is allowed, and also for the case that unbounded multiple-crossover recombination is allowed. The latter case also resolves a conjecture recently stated in (Huson and Klopper, 2007) in the context of reticulation networks. Although the conjecture from Gusfield and Bansal (2005) is disproved in general, we show that the answer to the conjecture is yes in several natural special cases, and establish necessary combinatorial structure that counterexamples to the conjecture must possess. We also show that counterexamples to the conjecture are rare (for the case of single-crossover recombination) in simulated data.

Algorithms to Distinguish the Role of Gene-Conversion from Single-Crossover Recombination in the Derivation of SNP Sequences in Populations

Meiotic recombination is a fundamental biological event and one of the principal evolutionary forces responsible for shaping genetic variation within species. In addition to its fundamental role, recombination is central to several critical applied problems. The most important example is "association mapping" in populations, which is widely hoped to help find genes that influence genetic diseases (Carlson et al., 2004; Clark, 2003). Hence, a great deal of recent attention has focused on problems of inferring the historical derivation of sequences in populations when both mutations and recombinations have occurred. In the algorithms literature, most of that recent work has been directed to single-crossover recombination. However, gene-conversion is an important, and more common, form of (two-crossover) recombination which has been much less investigated in the algorithms literature. In this paper, we explicitly incorporate gene-conversion into discrete methods to study historical recombination. We are concerned with algorithms for identifying and locating the extent of historical crossing-over and gene-conversion (along with single-nucleotide mutation), and problems of constructing full putative histories of those events. The novel technical issues concern the incorporation of gene-conversion into recently developed discrete methods (Myers and Griffiths, 2003; Song et al., 2005) that compute lower and upper-bound information on the amount of needed recombination without gene-conversion. We first examine the most natural extension of the lower bound methods from Myers and Griffiths (2003), showing that the extension can be computed efficiently, but that this extension can only yield weak lower bounds. We then develop additional ideas that lead to higher lower bounds, and show how to solve, via integer-linear programming, a more biologically realistic version of the lower bound problem. We also show how to compute effective upper bounds on the number of needed single-crossovers and gene-conversions, along with explicit networks showing a putative history of mutations, single-crossovers and gene-conversions. Both lower and upper bound methods can handle data with missing entries, and the upper bound method can be used to infer missing entries with high accuracy. We validate the significance of these methods by showing that they can be effectively used to distinguish simulation-derived sequences generated without gene-conversion from sequences that were generated with gene-conversion. We apply the methods to recently studied sequences of Arabidopsis thaliana, identifying many more regions in the sequences than were previously identified (Plagnol et al., 2006), where gene-conversion may have played a significant role. Demonstration software is available at www.csif.cs.ucdavis.edu/~gusfield.

Isolation and Characterization of a Dominant Negative Mutant of Bacillus subtilis GTP-binding Protein, YlqF, Essential for Biogenesis and Maintenance of the 50 S Ribosomal Subunit

The circularly permuted GTPase YlqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of YlqF (YlqFDeltaN10) inhibits cell growth even in the presence of wild-type YlqF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqFDeltaN10 acts as a competitive inhibitor of wild-type YlqF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqFDeltaN10-overexpressing cells and in YlqF-depleted cells, suggesting that YlqFDeltaN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFDeltaN10-overexpressing and YlqF-depleted cells more strongly enhanced the GTPase activity of YlqF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that YlqF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.

Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1.

We improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by combining electroporation with a top agar method. Transformation was also improved when a disruptant of a putative type I restriction endonuclease (tll2230) was used as recipient cells. In particular, some constructs, with which wild type has never been transformed, were successfully integrated into the tll2230-disruptant. Single-crossover recombination was detected more frequently than the double-crossover recombination. In accordance with the presence of all the homologs of pil genes in Synechocystis sp. PCC 6803, we found that T. elongatus is naturally transformable with exogenous DNA.

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.