GENOME ANALYSIS OF BURKHOLDERIA CEPACIA AC1100
William Hendrickson, Anette Hübner, Andrew Kavanaugh-Black and Reva Edelstein
Dept. of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612
SUMMARY
Burkholderia cepacia is an important organism in bioremediation of environmental pollutants and it is also of increasing interest as a human pathogen. The genomic organization of B. cepacia is being studied in order to better understand its unusual adaptive capacity and genomic plasticity. We are studying the diversity of chromosome number and size, and genome organization among different B. cepaciaisolates. Large replicons of several environmental and clinical isolates range in size from 0.3 to 5.3 million bp and vary in number from two to five. Strain AC1100 is of interest because it was isolated after selection for degradation of the herbicide 2,4,5-T. This strain contains four replicons of 0.34, 0.53, 2.7 and 4.0 Mbp. Genes coding for the enzymes required for the first step and for late steps in the degradative pathway were previously cloned. We have mapped the tft A gene to the 0.34 Mbp replicon and the tftE-H cluster to the 0.53 Mbp replicon. The data suggest that the 2,4,5-T pathway may have evolved by separate translocation events into each of the replicons. In the absence of 2,4,5-T, the genes are rapidly lost, most often by deletion and translocation events. Transposon mutagenesis has been used to create amino acid auxotrophs as well as new 2,4,5-T mutants to identify the remaining genes of the pathway. Using a cosmid bank of AC1100, a genetic and physical map of the strain is being constructed that can be used to compare B. cepacia strains in more detail.
IS elements containing outwardly oriented promoters were isolated from AC1100 using a promoter trap plasmid system. PCR analysis of the transposition products showed that two IS elements were responsible for most events. These elements could drive high level gene expression from as much as 600 base-pairs upstream of the gene. The IS elements are found near the tft genes, suggesting that they may play a role in the evolution of the pathway as well as the instability of the genes.
Key words: Genome mapping, 2,4,5-T, Burkholderia cepacia, insertion element
INTRODUCTION
Burkholderia (formerly Pseudomonas) cepacia is important in environmental microbiology and bioremediation of numerous toxic compounds (Lessie and Gaffney, 1986; Bhat et al., 1994; Shields and Reagin, 1992; Shields et al., 1995). It is also of increasing importance as a pathogen in cystic fibrosis and nosocomal infections (Mahenthiralingam et al., 1995; Sun et ali., 1995; Smith et al., 1993). B. cepaciaappear to possess highly plastic genomes with tremendous adaptive capacity. It is unclear how this capacity evolves and is maintained, but it has been proposed that genes are transferred laterally among Burkholderia and other genera, and that new metabolic capabilities are produced by integration of genes as well as modification of existing pathways for degradation of toxic compounds. Although there has been little data regarding the genetics and genome structure of B. cepacia, insertion elements have been implicated in the evolution of metabolic pathways and in plasmid and chromosomal reaarangements of various strains.
The strain AC1100 has acquired, during prolonged selection in continuous culture, genes for the utilization of the herbicide 2,4,5-T (Kilbane et al., 1982; Kellogg et al., 1981). Thus, AC1100 represents a rapidly evolved organism that appears to have acquired genes originally present in a consortium. Genes at two loci specifying a portion of this pathway were previously cloned, sequenced and the encoded enzymes characterized (Danganan et al., 1994; Daubaras et al., 1995).
It has long been assumed that all bacterial genomes consist of a single chromosome; however, recent data from physical mapping efforts have shown that several genera, including Burkholeria, contain multiple replicons (Suwanto and Kaplan, 1989; Allardet-Servent et al., 1993; Michaux et al., 1993; Zuerner et al., 1993; Cheng and Lessie, 1994). We are using physical mapping techniques to determine the overall structure of B. cepacia genomes, the distribution of insertion elements and the location of important catabolic genes.
MATERIALS AND METHODS
Bacterial Strains and Plasmids. The Escherichia coli strain JM109 was used for isolating plasmid DNA. Burkholderia cepacia strains AC1100, which metabolizes 2,4,5-T (Kilbane et al., 1982), and PT88, a transposon induced mutation of AC1100 (Sangodkar et al., 1988), were provided by A. Chakrabarty. B. cepacia 249 (ATCC17616) and 249-2, a spontaneous lysine auxotroph, were provided by T. Lessie (Gaffney and Lessie, 1987). N2P5 is a PAH degrader provided by S. Lantz and R. Devereaux. Clinical B. cepacia isolates B.c. 1, 2 and 3 were isolated from cystic fibrosis patients, and were provided by K. Williams and D. Allison. Plasmid pKS200 contains a 4.2 Kb portion of the tftE-H gene cluster and pUC119HBdE contains the complete tftE-H cluster cloned into pUC119 (Daubaras et al., 1995); pCD206 contains the tftA,B genes (Danganan et al., 1994); and pKT240 is an RSF1010 replicon with Km and Ap resistances and a promoterless Sm gene (Bagdasarian et al., 1983).
Culture conditions. Antibiotics used included 80 µg/ml Kanamycin (Km) and 20 µg/ml Streptomycin (Sm) for E. coli; 800 µg/ml Streptomycin and 150 µg/ml carbenicillin were used for B. cepacia isolates. Wild type B. cepacia AC1100 was grown in basal salt medium (BSM) containing 0.1% (w/v) 2,4,5 trichlorophenoxyacetic acid (2,4,5-T, Aldrich) (Kilbane et al., 1982). The other B. cepaciastrains were grown in BSM supplemented with 0.01% (w/v) yeast extract and 0.5% (w/v) glucose (BSMYEG). All Burkholderia strains were incubated at 30C. B. cepacia were plated on Pseudomonasisolation agar (PIA, Difco) supplemented with the appropriate carbon source.
Pulsed-Field Gel Electrophoresis. For preparation of agarose plugs containing chromosomal DNA, the high-molecular-weight DNA was prepared from overnight cultures as described (Cheng and Lessie, 1994) using commercially available disposable plug molds (Bio-Rad). Plugs used in separation of intact replicons were soaked in 50% glycerol, frozen at -80C and thawed at 37C. Glycerol was removed by washing the plugs in TE. For restriction enzyme digestion, the agarose plugs were immersed for 15 min. on ice in the reaction buffer (100 ml per agarose plug) recommended by the suppliers before addition of the restriction enzyme (5-10 units per plug). Following preincubation on ice for 1 hr, the samples were transferred to the appropriate reaction temperature and incubated for at least 3 hr. Following restriction digestion, the agarose slices were washed in TE before their transfer to the sample wells of agarose gels containing 0.8%, 1% or 1.2% (w/v) FastLane agarose (FMC BioProducts) and 0.5X TBE buffer (45 mM Tris-borate, 1 mM EDTA). The samples were resolved electrophoretically using a contour-clamped homogenous electric field (CHEF) apparatus (Bio-Rad DRII). The parameters for electrophoresis were set dependent on the range of molecular weights to be separated and were as follows:
| Size range (kb) | Agarose conc. (%) | Time (hr) | Pulse (sec) | Volts (V) |
| <400 | 1.2 | 16 | 5-40 | 200 |
| 150-1500 | 1 | 22 | 50-90 | 200 |
| 1500-5000 | 0.8 | 170 | 500-3400 | 50 |
Gels were stained in ethidium bromide and photographed using a UV photoimager system. The sizes of whole replicons and restriction fragments were estimated by using chromosomal DNA of Hansenula wingei and Saccharomyces cerevisiae or lambda phage concatamers as marker DNA.
Preparation of Probe DNA, Southern Transfer and Hybridization. All general methods were as described (Sambrook et al., 1989). DNA restriction fragments (tft-genes, IS931) or PCR products were used as probes. The primers used for the PCR of the 16S rRNA gene were from the sequence of B. cepacia17616 (R. Devereaux, personal communication): 5'-AGAGTTTGATCCTGGCT
CAG and 5'-ACGGCTACCTTGTTACGACTT. The DNA was labeled by the random priming labeling technique (New England Biolabs NBlot kit). Radio labeling of excised PFGE macro-restriction fragments was performed as described (Chen and Widger, 1993). Excised fragments were melted at 80C, diluted, denatured in a boiling water bath for 5 minutes and then cooled to 65C. Prewarmed reaction components were added and the reaction was carried out at room temperature for 2 hours.
After staining with ethidium bromide, gels were exposed to UV irradiation at 600 mJ/cm2, DNA was transferred to Nytran (Schleicher and Schuell) membranes using a TurboBlotter (Schleicher and Schuell) for 6 hours. DNA was fixed to the membranes by UV cross linking (1200 mJ/cm2). Prehybridization was carried out as described (Sambrook et al., 1989) with the exception that 10% (w/v) dextran sulfate (Pharmacia) was added. Hybridization was carried out over night at 65C in the same solution.
IS Elements. The promoter probe vector pKT240 containing a promoterless streptomycin resistance gene was transformed into B. cepacia AC1100 by electroporation. Carbenicillin resistant transformants were selected on PIA plates containing 150 µg/ml Cb. Single colonies were picked and grown in BSM glucose (BSMG) containing the same amount of Cb and then plated on PIA plates containing 800 µg/ml streptomycin to select for transposition events. Plasmid DNA was prepared from the clones using the CTAB-DNA precipitation method (Del-Sal et al., 1987) and then electroporated into JM109. Bacterial colonies were lysed by microwaves, and insertion events were characterized directly by PCR (Kilger and Schmid, 1994).
Transposon Mutagenesis. The transposon Tn5-751S (Chen and Widger, 1993) confers resistance to kanamycin and contains a unique Swa I site. Tn5-751S was transferred to the broad-host range "suicide" vector pSUP202 from pTGL166. The new replicon, pAB3, contains Tn5-751S inserted into the pSUP202 tetr gene. pAB3 was mated into AC1100 by triparental mating using the conjugation helper plasmid pRK2013. Kanamycin resistant AC1100 colonies were selected on Pseudomonas Isolation Agar (PIA). The library was screened for growth on 0.1% 2,4,5-T/Basal Salts Media supplemented with sub-optimal concentrations of glucose or glucose and yeast extract. Putative 2,4,5-T- mutants were further screened for growth on Glucose/Basal Salts Media (BSMG; 0.2% w/v glucose) supplemented with 0.1% Casamino acids.
RESULTS AND DISCUSSION
Burkholderia cepacia Replicons. Pulsed-field gel electrophoresis has been used to determine the sizes of bacterial chromosomes and to create physical maps of rare-cutting restriction enzyme sites. Linear DNA up to 6 million base-pairs (Mbp) can be resolved. We have used this technique to measure chromosome sizes of several Burkholderia cepacia isolates. Gels were run in a CHEF apparatus for 22 hr to obtain accurate size data for the smaller replicons of 0.15 to 1.5 mb (Table 1). Data for larger replicons were obtained with gels run for 170 hr. Circular DNA does not run into these gels, so bands are only obtained for those molecules that have been linearized by a single random break. To increase the yield of DNA entering the gels from the agar plugs used for cell lysis, a freeze-thaw step was introduced. Glycerol was soaked into the agar plugs, the plugs were frozen in dry ice-ethanol, thawed at 37C, and the glycerol removed. We found a substantial increase in the DNA in the gel without a substantial increase in background smear of highly broken DNA. The genome of AC1100 has five replicons of 4, 2.7, 0.53, 0.34 and 0.15 megabases that are distinguished by pulsed-field gel electrophoresis (Table 1). The replicons, from the largest to the smallest, have been designated 1 to 5. The total genome size is 7.7 mb. Restriction enzyme mapping (see below) demonstrates that these DNA species do not result from breaks in larger molecules, but are independent circular replicons.
The replicon number and the sizes vary considerably among the B. cepacia strains studied. On the basis of 16S rRNA homology, strain N2P5 is quite similar to AC1100, but the replicon sizes are very different (Table 1). N2P5 has the largest total genome size of 9.3 mb, more than 20% larger than AC1100. The environmental isolate 10661 also has a large genome with three replicons of 1.6 to 3.7 Mbp. This isolate was the only one that did not appear to harbor any smaller replicons less than 1.6 Mbp. Multiple chromosomes of Burkholderia were first demonstrated in strain 249 (ATCC17616) (Cheng and Lessie, 1994). Our size estimates are likely to be accurate, since our data for 249 are identical with those previously published. We also tested three clinical isolates from cystic fibrosis patients and found the same variation in replicon number and size (strains B.c. 1, 2 and 3 in Table 1). Although the variation in overall genome size is not notable, the sizes of individual replicons vary dramatically. We have obtained additional clinical isolates, and we will compare strains that have very different clinical outcomes and isolates obtained over time from the same patient to look for correlations of genome structure and clinical state.
Locations of tft Genes. B. cepacia AC1100 is capable of utilizing the defoliant 2,4,5-T as a sole source of carbon and energy. This capability is quickly lost in the absence of 2,4,5-T (Chatterjee et al., 1982). A metabolic pathway was suggested based on studies of intermediates produced by the parental and mutant strains (Sangodkar et al., 1988; Sangodkar et al., 1989). Two gene clusters involved in the catabolism of 2,4,5-T by AC1100 have been cloned and characterized. The tftA and tftB genes encode an oxygenase required for the first step, the conversion of 2,4,5-T to 2,4,5-TCP (Danganan et al., 1994). A second clone, containing genes designated tftEFGH, complements a transposon-induced mutation (PT88) unable to grow on 2,4,5-T (Daubaras et al., 1995). The PT88 mutant accumulates 5-chloro-1,2,4-trihydroxybenzene (oxidized to 5-chloro- hydroxyhydroquinone), which is thought to be an intermediate in the lower portion of the pathway (Figure 1). Based on amino acid homology, the activities of tftH and tftE genes have been suggested (Figure 1) (Daubaras et al., 1995). The two sets of tft genes are flanked by insertion elements, IS931 and IS932, which may have been involved in the transfer into B. cepacia. We looked at the chromosomal location of the cloned genes by performing Southern blot hybridization to whole chromosomes separated by pulsed-field gel electrophoresis. The tftA,B genes are located on the 0.34 Mbp replicon 4, whereas the tftE-H cluster is located on the 0.53 Mbp replicon 3. The separate locations of genes for the upper and lower portions of the pathway and their association with IS elements is consistent with the possibility that the pathway was assembled by multiple insertional events. Alternatively, during the evolution of AC1100, a single gene cluster may have rearranged and split apart after a single initial genetic exchange. It will be interesting to determine the chromosomal locations of the remaining genes of the pathway once they are cloned.
We are now mapping the locations of the IS elements and assessing whether specific portions of the genome (specific chromosomes?) are involved in deletion and insertion events. In at least one case this seems to be true. Strain PT88 was thought to be a transposon insertion mutation of the tftE gene cluster. We have shown, however, that PT88 has undergone considerable rearrangement, including deletion of the tftE gene cluster. Replicon 3 has increased in size by 30 kb and Replicon 4 has been deleted of 30 kb (Table 1). DNA of the cloned tftA,B and tftE-H genes was used to probe Southern blots of whole PT88 replicons. Surprisingly, the tftA,B genes have translocated from replicon 3 to replicon 4. In the presence of 2,4,5-T, PT88 rapidly mutates to become tftA,B deficient. These mutations are easily detected on plates by a failure to produce the pink colored CHQ intermediate that is accumulated by PT88 cells. Colonies that failed to produce pink color on 2,4,5-T plates and in liquid media were isolated. In the tftA,B mutants, a 300Kb deletion of replicon 3 had occurred in each of three candidates studied. Thus, we could not find a small "cassette" capable of moving these catabolic genes, but the genome analysis allowed us to identify a larger rearrangement that occurs at high frequency. These events may be similar to the ones that established the genes in AC1100 initially, and they may be responsible for the rapid loss of 2,4,5-T catabolic potential when AC1100 is used in the field. Since the predominant rearrangements appear to be specifically directed towards certain chromosome locations, there may be elements such as Insertion Sequences causing hyper-recombination.
We have begun to assemble a macrorestriction map of AC1100 using the enzymes, Swa I, Pac I, Pme I, Ceu I and Spe I. Such a map will afford us a more detailed comparison of chromosome structure with the previously mapped strain 249, and the map will be useful in further analysis of chromosomal rearrangements. Restriction enzyme Ceu I has been shown to cut within 23s rRNA sequences of many Gram negative organisms (Liu et al. 1992); therefore, the enzyme can be used to map the rRNA genes. A Ceu I digest of AC1100 produces three large DNA fragments and three small fragments of 500, 300 and 200 kb. This result suggest that there are at least 6 rRNA genes. A 16S rRNA gene probe was made by PCR using AC1100 template and highly conserved primers. Using the probe, the rRNA genes of AC1100 were found only in the two largest replicons. DNA from individual replicons and restriction fragments was excised from pulsed-field gels, labeled and used as probes for Southern blot hybridizations. Swa I and CeuI digested DNA fragments were first assigned to the appropriate replicons, and then fragment linkages determined. Using this method, physical maps for replicons 1 and 2 were constructed and show that they are independent, circular molecules.
Transposon Mutagenesis. Transposon mutants of AC1100 would be useful for obtaining genetic markers such as amino acid auxotrophs to create a combined genetic/physical map, as well as for identifying and cloning additional genes involved in 2,4,5-T metabolism. AC1100 was mutagenized with a transposon, Tn5-751S, containing a Swa I site for physical mapping and resistance genes for both kanamycin and trimethoprim. The original vector, pTGL166, is a broad-host range replicon containing a temperature sensitive replication mutation; however we could not select against replication of the vector since AC1100 is thermosensitive and will not grow at temperatures at or above 40C. Therefore, we transferred Tn5-751S to the "suicide" vector pSUP202 since it is mobilizable but non-replicating in most gram-negative bacteria. Tn5-751S was transferred to pSUP202 by cotransformation of DH5a with both pTGL166 and pSUP202, then selecting for loss of the pTGL166 replicon by growth at 42C while maintaining selection for the Tn5-751S transposon and the pSUP202 replicon.
The resulting plasmid, pAB3, was mated into AC1100 by triparental mating and kanamycin resistant AC1100 colonies selected. Approximately 10,000 independent transposon mutants were obtained. The library was screened for growth on mannitol/Basal Salts Media supplemented with suboptimal concentrations of vitamin free Casamino Acids to allow initial growth of all colonies. Thirty seven putative amino acid auxotrophs that remained as micro colonies on these plates were screened further for growth on glucose/Basal Salts Media without Casamino acids supplementation. Of these, thirty three which required BSMG supplemented with either Yeast Extract or Casamino acids were selected for further study. Subsequently 5 of the 33 were found to replicate on Citrate/Basal Salts Media, leaving 28 putative amino acid auxotrophs. The specific auxotrophic mutation responsible for the casamino acid requiring phenotype was determined by plating on BSMG with various combinations of amino acids. Ten different amino acid auxotrophs were identified: ser, ilv, pro, phe, tyr, arg, met, trp, leu and thr. The corresponding cosmid clones are being isolated using a previously constructed AC1100 BamH I/pCP13 cosmid library. Twenty three auxotrophic transposon mutants of AC1100 were complemented by mating in the cosmid library. Four complementing clones have been isolated and transferred to E. coli: phe, tyr, met and trp. The clone containing the phe genes was tested on four independent phe mutants and all were complemented. The library of Tn5-751 mutated AC1100 was also screened for the recA- phenotype by isolating MMS-sensitive cells, and complementing clones were obtained. We will use these clones as probes to map the amino acid genes and recA to specific replicons and restriction fragments.
A modification of the selection procedure described for the isolation of amino acid auxotrophs was used to isolate AC1100/Tn5-751S 2,4,5-T mutants. The library was screened for growth on 2,4,5-T/Basal Salts Media supplemented with sub-optimal concentrations of glucose or glucose and yeast extract. One hundred nine putative 2,4,5-T- mutants were further screened for growth on glucose/Basal Salts Media supplemented with casamino acids. Ninety seven mutants exhibited limited or no growth on 2,4,5-T/Basal Salts Media as sole source of carbon, yet grow well on BSMG or BSMC and have been selected for further study. The mutants were screened for complementation with the two clones of 2,4,5-T degradation genes. These two operons, contained in the vectors pUS1010 (Sangodkar et al., 1988) and pRHC89 (Danganan et al., 1994) are required for growth on 2,4,5-T as sole carbon source. Given the large number of 2,4,5-T-mutants isolated, we expect that the map of 2,4,5-T degradation genes has been saturated, and if there are unidentified genes necessary for the growth of AC1100 on 2,4,5-T we should be able to clone and map them on the AC1100 genome. Most of the mutants were complemented by the clones containing the previously identified tft genes. Ten mutants that failed to complement are being studied further. Two mutants produce a pale pink color, suggesting a block at the distal part of the pathway and accumulation of chloro-hydroxyhydroquinone, but these mutants produce less color than PT88. The product will be identified by HPLC. We tested each candidate for production of trichlorophenol, the first step in the pathway, and for dechlorinase activity, the second and third steps. Mutant AB10T is not able to convert 2,4,5-T to 2,4,5-TCP; therefore it is likely defective in the 2,4,5-T oxygenase (Figure 1). Since the mutant was not complemented by the tftA,B clone, it must have undergone additional mutation in the pathway or it is altered in a regulatory function. Mutants AB9T and AB204T produce TCP but have no dechlorinase activity, whereas AB181T has partial dechlorinase activity. These mutations are likely in the TCP or DCHQ oxygenase genes or related regulatory functions (Figure 1). The strains are now being used to screen for clones expressing these activities.
Insertion Elements in B. cepacia AC1100. Two putative insertion elements, IS931 and IS932, were previously found associated with both of the cloned tft gene clusters (Haugland et al., 1990; Tomasek et al., 1989). Although IS932 was not characterized, IS931 was sequenced and shown to transpose and activate adjacent genes. A probe containing IS931 sequence was produced by PCR and hybridized to the AC1100 replicons. IS931 was found to be present in multiple copies in AC1100 on all 4 large replicons. It was absent from B. cepacia strains 17616 (249), 25416, and N2P5 by Southern hybridization under stringent conditions with EcoR I-digested genomic DNA of these strains. Also, the tftA and tftD genes, present in AC1100, are lacking in N2P5 as probed under stringent conditions. A number of IS elements have been characterized in strain 249 (Lessie et al., 1990; Wood et al., 1991), but hybridizations with probes containing these elements indicate that they are not present in AC1100.
We are using a "promoter trap" vector to observe IS activity and to look for additional IS elements with promoter activity. The vector has a promoterless streptomycin resistance gene that can be driven by promoters from elements inserted upstream of the translational start site (Figure 2). We developed conditions for electroporation of the vector, pKT240, and it was electroporated into AC1100, and cells were plated for high str resistance at 800 g/ml. Several techniques for screening the candidates were tried in an effort to develop a simple accurate method for isolating insertions, and PCR proved easy and reliable. Primers were synthesized for the vector region between the str gene and a kan marker 1 kb upstream (Figure 2). An additional primer corresponding to the promoter region of IS931 was also made. Plasmid from each candidate was transformed into E. coli and plated for amp resistance. Colonies were picked, placed in a tube and microwaved, followed directly by PCR. Primer concentrations were adjusted and 10% DMSO added to obtain maximal product from large inserts. All the primers were used simultaneously so that any insertions were detected and the presence of IS931 was also seen in the same sample. Since IS932 was never characterized, we could not use PCR to detect it. All candidates that contained IS elements but were negative for IS931 were characterized by restriction analysis of the PCR products.
Of 50 candidates that were fully characterized, 24 had IS elements, 13 are IS931, 11 were identical to each other and are likely to be IS932 based on size and restriction pattern (Table 2). One insertion is a small fragment of 400 bp which retains the promoter activity of an IS931 element. It could have resulted from insertion of one IS element into another and then improper excision removing all but one end of the IS. This construct provides an interesting example of how a promoter could be "evolved" from an insertion element with little remaining evidence of the original insertion event. By measuring the size of the PCR product, an accurate measure of the distance of the IS element from the str gene start site was obtained. High level transcriptional activity was observed with insertions up to 600 bp away. Using another insertion trap vector containing the sacB gene, we find that IS931 and IS932 produce most, if not all, the insertion events in this strain even when promoter activity is not selected. These insertion elements are highly active and their transposition and recombination may drive much of the chromosomal instability of AC1100.
ACKNOWLEGEMENTS
We thank C. Danganan, D. Daubaras, A. Chakrabarty, T. Lessie, S. Lantz, R. Devereaux, K. Williams and D. Allison for plasmids and strains. This work was supported by U.S. EPA Cooperative agreement CR822632.
REFERENCES
Allardet-Servent A S, Michaux-Characon S, Jumas-Bilak E, Karayan L, Ramuz M (1993) Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J. Bacteriol. 175:7869-7874.
Bagdasarian M M, Amann E, Lurz R, Ruckert B, Bagdasarian M (1983) Activity of the hybrid trp-lac(tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26:273-282.
Bhat M A, Tsuda M, Horiike K, Nozaki M, Vaidyanathan C S, Nakazawa T (1994) Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichloro-phenoxyacetate from Pseudomonas cepacia CSV90. Appl. Environ. Microbiol. 60:307-312.
Chatterjee D K, Kilbane J J, Chakrabarty A M (1982) Biodegradation of 2,4,5-trichlorophenoxyacetic acid in soil by pure culture of Pseudomonas cepacia.. Appl. Env. Micro. 44:514-516.
Chen H -P, Widger W R (1993) Physical Genome Map of the Unicellular Cyanobacterium Synechococcussp. Strain PCC 7002 . J. Bacteriol. 175:5106-5116.
Cheng H -P, Lessie T G (1994) Multiple replicons constituting the genome of Pseudomonas cepacia17616. J. Bac. 176:4034-4042.
Danganan C E, Ye R W, Daubaras D L, Xun L, Chakrabarty A M (1994) Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100. Appl. Env. Micro. 60:4100-4106.
Daubaras D L, Hershberger C D, Kitano K, Chakrabarty A M (1995) Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100. Appl. Environ. Microbiol. 61:1279-1289.
Del-Sal G, Manfioletti G, Schneider C (1987) The CTAB-DNA Precipitation Method: A Common Mini-Scale Preparation of Template-DNA from Phagemids, Phages or PLasmids Suitable for Sequencing. Biotechniques 7:514-519.
Gaffney T D, Lessie T G (1987) Insertion-sequence-dependent rearrangements of Pseudomonas cepaciaplasmid pTGL1. J. Bacteriol. 169:224-230.
Haugland R A, Sangodkar U M X, Chakrabarty A M (1990) Repeated sequences including RS1100 from Pseudomonas Cepacia AC1100 function as IS elements. Mol. Gen. Genet. 220:222-228.
Kellogg S T, Chatterjee D K, Chakrabarty A M (1981) Plasmid-assisted molecular breeding: new technique for enhanced biodegradation of persitent toxic chemicals. Science 214:1133-1135.
Kilbane J J, Chatterjee D K, Karns J S, Kellogg S T, Chakrabarty A M (1982) Biodegradation of 2,4,5-trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia. Appl. Env. Micro. 44:72-78.
Kilger C, Schmid K (1994) Rapid characterisaztion of bacterial clones by microwave treatment and PCR. Trends Genet. 10:149.
Lessie T G, Wood MS, Byrne A, Ferrante A (1990) Transposable gene-activating elements in Pseudomonas cepacia, p. 279-291. In: S. Silver, A. M. Chakrabarty, B. Iglewski, and S. Kaplan (eds.), Pseudomonas: Biotransformations, Pathogenesis and Evolving Biotechnology . American Society for Microbiology, Washington.
Lessie, T G, Gaffney T (1986) Catabolic potential of Pseudomonas cepacia, p. 439-481. In: J.R. Sokatch and L.N. Ornston (eds.), The Bacteria, vol. 10. The Biology of Pseudomonas. Academic Press, Orlando, FL.
Liu S -H, Hessel A, Sanderson K E (1992) Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA in Salmonella spp., E. coli, and other bacteria. Proc. Natl. Acad. Sci. ,USA 90:6874-6878.
Mahenthiralingam E, Campbell M, Speert D P (1995) Burkholderia cepacia in cystic fibrosis. N. Engl. J. Med. 332:819.
Michaux S, Paillisson J, Carles-Nurit M-J, Bourg A, Allardet-Servent A, Ramuz M (1993) Presence of two independent chromosomes in the Brucella melitensis 16M genome. J. Bacteriol. 175:701-705.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York.
Sangodkar U M X, Chapman P J, Chakrabarty A M (1988) Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T in Pseudomonas cepacia AC1100. Gene 71:267-277.
Sangodkar U M X, Aldrich T L, Haugland R A, Johnson J, Rothmel R K, Chapman P J, Chakrabarty A M (1989) Molecular basis of biodegradation of chloroaromatic compounds. Acta Biot. 9:301-316.
Shields M S, Reagin M J (1992) Selection of a Pseudomonas cepacia strain constitutive for the degradation of trichloroethylene. Appl. Environ. Microbiol. 58:3977-3983.
Shields M S, Reagin M J, Gerger R R, Campbell R, Somerville C (1995) TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4. Appl. Env. Micro. 61:1352-1356.
Smith D L, Gumery L B, Smith E G, Stableforth D E, Kaufmann M E, Pitt T L (1993) Epidemic of Pseudomonas cepacia in an adult cystic fibrosis unit: evidence of person-to-person transmission. J. Clin. Microbiol. 31:3017-3022.
Sun L, Jiang R Z, Steinbach S, Holmes A, Campanelli C, Forstner J, Sajjan U, Tan Y, Riley M, Goldstein R (1995) The emergence of a highly transmissible lineage of cbl+ Pseudomonas (Burkholderia) cepaciacausing CF centre epidemics in North America and Britain. Nature Med. 1:661-666.
Suwanto A, Kaplan S (1989) Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J. Bacteriol. 171:5850-2859.
Tomasek P H, Frantz B, Sangodkar U M X, Haugland R A, Chakrabarty A M (1989) Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia. Gene 76:227-238.
Wood M S, Byrne A, Lessie T G (1991) IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia. Gene 105:101-105.
Zuerner R L, Herrmann J L, Saint Girons I (1993) Comparison of genetic maps for two Leptospira interrrogans serovars provides evidence for two chromosomes and intraspecies heterogeneity. J. Bacteriol. 175:5445-5451.
Figure 1. Mutations in the proposed 2,4,5-T catabolic pathway of B. cepacia AC1100.
Figure 2. Vector for trapping Insertion Elements with promoter activity. Filled rectangle indicates inserted IS element; arrows show positions of PCR primers used in locating IS elements.