Improved degradation of 4-chlorobiphenyl, 2,3-dihydroxybiphenyl, and catecholic compounds by recombinant bacterial strains

ThepcbC gene encoding (4-chloro-)2,3-dihydroxybiphenyl dioxygenase was cloned from the genomic DNA ofPseudomonas sp. P20 using pKT230 to construct pKK1. A recombinant strain,E. coli KK1, was selected by transforming the pKK1 intoE. coli XL1-Blue. Another recombinant strain,Pseudomonas sp. DJP-120, was obtained by transferring the pKK1 ofE. coli KK1 intoPseudomonas sp. DJ-12 by conjugation. Both recombinant strains showed a 23.7 to 26.5 fold increase in the degradation activity to 2,3-dihydroxybiphenyl compared with that of the natural isolate,Pseudomonas sp. DJ-12. The DJP-120 strain showed 24.5, 3.5, and 4.8 fold higher degradation activities to 4-chlorobiphenyl, catechol, and 3-methylcatechol than DJ-12 strain, respectively. The pKK1 plasmid of both strains and their ability to degrade 2,3-dihydroxybiphenyl were stable even after about 1,200 generations.     

Properties of plasmids from Methylomonas clara

Three independently isolated clones of the obligate methylotrophic bacterium Methylomonas clara (ATCC 31226) were tested for their plasmid content. Strains B45-BE-2 and B45-BE-3 were shown to harbor one class of plasmid molecules each, with molecular sizes of 28 MDa (46 kb) and 10 MDa (16 kb), respectively. Strain B45-BE-1 does not contain extrachromosomal DNA. Restriction maps of plasmid pBE-2 from strain B45-BE-2 and of plasmid pBE-3 from strain B45-BE-3 were established and the smaller plasmid pBE-3 shown to be a linear deletion derivative of plasmid pBE-2. Two other deletions were characterized. When subcloned into the Escherichia coli vector pBR322, plasmid pBE-3 was unable to complement for the polymerase A dependence of the pBR322 replicon. Additional experiments indicate a general failure of the M. clara specific replicon to function in E. coli.     

The ORF1 of the gentamicin-resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5'-phosphosulphate kinase

The gentamicin-resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine-5′-phosphosulphate (APS) kinase. APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3′-hydroxyl group of APS. The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3′-hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E. coli APS kinase. In extracts of pseudomonad cells we have also detected a higher molecular mass (70 kDa) protein that cross-reacts with an anti-E. coli APS kinase antibody. This cross-reactive protein is also present in Pseudomonas strains lacking the gentamicin-resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ-encoded high-molecular-weight APS kinase present in Rhizobium meliloti. Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E. coli, as is E. coli APS kinase. However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1 -encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.     

Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551

A replicon from one of an array of seven indigenous compatible plasmids ofBacillus megateriumQM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bpHindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3′ end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act intrans.A small region with similarity to theB. subtilischromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressedrepgene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of aB. megateriumreplicon.     

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.     

Molecular cloning of a chromosomal DNA region encompassing the dihydrofolate reductase gene ofStreptococcus pneumoniae

Natural competence ofStreptococcus pneumoniae was used to locate and enrich DNA restriction fragments, biologically active for transformation of thymidine-deficient to thymidine-proficient cells. Mutations in the dihydrofolate reductase gene are accompanied by resistance to the drug trimethoprim (Tp). A 6.5-kb region of the pneumococcal chromosome encompassing the dihydrofolate reductase gene has been cloned in plasmid pLS1.Escherichia coli mutants, resistant to Tp, became fully sensitive to the drug when they harbored the recombinant plasmid. The pneumococcaldfrA mutation has been mapped within a 500-bp DNA region.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

Cryptic plasmid pBf1 fromButyrivibrio fibrisolvens AR10: Its use as a replicon for recombinant plasmids

A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacteriumButyrivibrio fibrisolvens strain AR10, ampped with restriction endonucleases, and cleavage sites suitable for attachment toEscherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42°C or in 0.25 g ampicillin/ml, but growth in 50 g acridine orange/ml for three culture passages produced cured colonies at a frequency of <1%. Chimeric plasmids were constructed by combining pBf1 with theE. coli plasmid pUC18, in addition to the clindamycin resistance gene fromBacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene fromE. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.     

Host range of conjugation and replication functions of the Escherichia coli sex plasmid Flac. Comparison with the broad host-range plasmid RK2

The Escherichia coli conjugative plasmid Flac has a restricted host range, in that transfer to Pseudomonas aeruginosa is not detectable. The molecular basis for this host-range restriction was studied by a separate comparison of the replication and conjugation systems of Flac with those of the broad host-range plasmid RK2. The origin of transfer of Flac (oriT_F) was cloned onto a small RK2 replicon. The hybrid plasmid, pDG2906, could be transferred efficiently by both the Flac and RK2 conjugation systems to an E. coli recipient. The Flac conjugation system was able to transfer pDG2906 to P. aeruginosa, but only at a frequency of 10^?4 of that of the RK2 conjugation system. A second hybrid plasmid, containing the replication region of Flac with the transfer region of RK2, could not be established in P. aeruginosa. These results show that Flac is able to mediate low frequency transfer to P. aeruginosa, and that the lack of replication in Pseudomonas is ultimately responsible for the restricted host range.     

Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.     

The use of plasmid R1162 and derivatives for gene cloning in the methanol-utilizing Pseudomonas AM1

Summary A physical map for plasmid R1162 (Sm, Su, IncP4) was constructed. Neither EcoRI, PstI nor EcaI cut within a region essential for replication, mobilization or streptomycin resistence. Plasmid R1162 can replicate in E. coli as well as in Pseudomonas species and shows a strong dependence for DNA polymerase I in E. coli. By RP4 induced mobilization, R1162 can be transferred from E. coli to Pseudomonas AM1. A hybrid plasmid pFG7 (MW=8.4×10⁶, Sm, Su, Ap, Tc) was constructed between pBR322 and R1162, which allows the selection of hybrid plasmids by insertional inactivation with the restriction enzymes HindIII, BamHI, SalI, ClaI. Transformation of E. coli SK1592 with EcaI-cut and ligated R1162-DNA and Pseudomonas AM1-DNA and subsequent mobilization of the hybrid plasmids into Pseudomonas AM1/M15a (methanol dehydrogenase^-) led to the isolation of Pseudomonas AM1/M15a colonies, which could grow on methanol again. Back-conjugation into E. coli SK1592, subsequent mobilization studies and plasmid analysis suggests that the gene for Pseudomonas methanol dehydrogenase has been cloned in this vector.     

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus–Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.     

Evidence for plasmid-mediated resistance ofPseudomonas putida to hexahydro-1,3,5-triethyl-s-triazine

Resistance to the industrial biocide hexahydro-1,3,5-triethyl-s-triazine (HHTT) by a strain ofPseudomonas putida was shown to be encoded by a 32.5-megadalton (Mdal) plasmid as evidenced: (a) by visualization of the plasmid DNA by agarose gel electrophoresis, (b) by the loss of HHTT resistance as well as the loss of the 32.5-Mdal plasmid upon curing with novobiocin, and (c) by conjugal concomitant transfer of HHTT resistance and the 32.5-Mdal plasmid by mating the novobiocin-cured HHTT-sensitive derivative with the HHTT-resistant strain. The 32.5 Mdal did not encode for heavy metal or antibiotic resistance, and it was shown not to be one of the degradative plasmids ofPseudomonas. The mechanism of HHTT resistance was not discerned from these studies.     

Biodegradation of malachite green by strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K9 and cloning of the <Emphasis Type="Italic">tmr2</Emphasis> gene associated with an IS<Emphasis Type="Italic">Ppu12</Emphasis>

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H⁺ antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.     

Cold tolerance of Pseudomonas sp. 30-3 isolated from oil-contaminated soil, Antarctica

Pseudomonas sp. 30-3 was enriched from oil-contaminated soil from Wright Valley, Antarctica using JP8 jet fuel as sole carbon source. This isolate exhibited tolerance to temperatures ranging from 0°C to 35°C when cultured in laboratory medium. In a freeze-thaw study, an 89% survival was observed when Pseudomonas sp. 30-3 was exposed to 4°C prior to freezing. PCR amplification of a 248-bp DNA fragment in Pseudomonas sp. 30-3 using capB-gene specific primers showed a 98% amino acid sequence homology with CapB of Pseudomonas fragi and 62% homology with CspA of Escherichia coli. Radiolabeling of total cellular proteins exhibited elevated expression of an 8-kDa protein at 4°C, which suggests that the CapB in Pseudomonas sp. 30-3 may play a pivotal role in survival and tolerance at cold and subzero temperatures. Tolerance to cold temperatures and the ability to degrade hydrocarbons by Pseudomonas sp. 30-3 provide support for the application of bioremediation for petroleum hydrocarbons in Antarctic soils.     

Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis

pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328. pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var. japonica. The replicon of pOM1 includes the ori region and the repA gene. The ori region, located upstream of the repA gene includes two sets of 31- and 13-bp direct repeats (DR), with AT-rich regions preceding each of the DRs. Two putative promoters of the repA gene were found connected with the DR regions. A 40-kDa protein was encoded by the repA gene and found essential for replication. Expression of the tetC gene is regulated by an Escherichia coli sigma(70)-like promoter and is dependent on the F. tularensis strain and its environment.     

Cloning of an endoglucanase gene fromPseudomonas fluorescens var.cellulosa intoEscherichia coli andPseudomonas fluorescens

Summary An endoglucanase chromosomal gene from the cellulolyticPseudomonas fluorescens var.cellulosa (NCIB 10462) was cloned inEscherichia coli. Chromosomal DNA was partially digested with the restriction enzymeEcoRI and ligated into the broad host-range, mobilizable plasmid pSUP104 that had been linearized with the same enzyme. After transformation ofEscherichia coli, and endoglucanase-positive clone was detected in situ by use of the Congo-red assay procedure. The endoglucanase gene on the recombinant plasmid pRUCL 100 was expressed in the non-cellulolyticPseudomonas fluorescens PF41. The DNA fragment carrying the gene was transferred to the plasmid pBR322, generating plasmids pRUCL150 and pRUCL151, and its restriction map was derived.Abbreviations CMC carboxymethylcellulose - EG endoglucanase - kb kilobase pairs - Mops 4-morpholinepropanesulfonic acid - Ap^r-s resistance-sensitivity to the antibiotic ampicillin - Cm^r-s resistance-sensitivity to the antibiotic chloramphenicol - Tc^r-s resistance-sensitivity to the antibiotic tetracycline - Sm^r-s resistance-sensitivity to the antibiotic streptomycin - Tp^r-s resistance-sensitivity to the antibiotic trimethoprim