Map of the IncP1β Plasmid pTSA Encoding the Widespread Genes (tsa) for p-Toluenesulfonate Degradation in Comamonas testosteroni T-2

The catabolic IncP1β plasmid pTSA from Comamonas testosteroni T-2 was mapped by subtractive analysis of restriction digests, by sequencing outwards from the tsa operon (toluenesulfonate degradation), and by generating overlapping, long-distance-PCR amplification products. The plasmid was estimated to comprise 72 ± 4 kb. The tsa region was found to be a composite transposon flanked by two IS1071 elements. A cryptic tsa operon was also present in the tsa transposon. Those backbone genes and regions which we sequenced were in the same order as the corresponding genes in resistance plasmid R751, and identities of about 99% were observed. Enrichment cultures with samples from four continents were done to obtain organisms able to utilize p-toluenesulfonate as the sole source of carbon and energy for aerobic growth. Most (15) of the 16 cultures (13 of them isolates) were obtained from contaminated sites and were attributed to three metabolic groups, depending on their metabolism of p-toluenesulfonate. The largest group contained the tsa transposon, usually (six of seven isolates) with negligible differences in sequence from strain T-2.     

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.     

Characterization of TsaR,an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR(His). TsaR(His) was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR(His) to be a monomer in solution. Overexpressed TsaR(His) bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K(D)) for the whole promoter region was about 0.9 micro M, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.     

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.     

Mutations of plasmid pBS286 blocking the initial stages of naphthalene oxidation induced by Tn5

A collection of Tn5 transposon Nah- mutants of the plasmid pBS286 was obtained. The insertion sites were localized and orientation of Tn5 determined. The mutants obtained were biochemically analyzed, the nah-region map of the plasmid being elaborated. Structural genes of the nah operon were shown to be organized similarly to those of the nah1 operon of the NAH7 plasmid discussed in the literature. The data obtained are in favour of the previously published information on the presence of elements operating the pBS286 plasmid. The results are given indicating a possibility of regulating the expression of catechol splitting meta-pathway genes with participation of products on early stages of naphthalene oxidation.     

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.     

Cloning and characterization of the mutated threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens

The entire threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens TLr156, which lacks threonine-mediated feedback inhibition of both aspartokinase I (AK I) and homoserine dehydrogenase I (HD I), was cloned on a multicopy plasmid pLG339. Hybrid plasmid pSK301 carried a 6.5-kb chromosomal DNA. Several derivatives of pSK301 with Tn1000 insertions were obtained. By examining the phenotypes and the physical maps of these plasmids, we could define the loci of the thrA(1)5A(2)5, thrB, and thrC genes. The thrA(1)5A(2)5 and thrC gene products were identified by the maxicell method as proteins with Mrs of 85,000 and 43,000, respectively. The thrA(1)5A(2)5 genes encode a single polypeptide similar to the thrA1A2 genes of Escherichia coli. Plasmid pSK301 was introduced into S. marcescens T-1112, in which both AK I and HD I are produced constitutively. The resulting transformant carried five to six copies of pSK301 per chromosome and produced the AK I and HD I enzymes at three to four times higher level than control strain T-1112[pLG339]. Strain T-1112[pSK301] produced four times higher levels of threonine than strain T-1112[pLG339], yielding about 35 mg of threonine per ml of a medium containing sucrose and urea.     

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn<Emphasis Type="Italic">MERI1</Emphasis>

Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.     

Selection of clc, cba, and fcb chlorobenzoate-catabolic genotypes from groundwater and surface waters adjacent to the Hyde park, Niagara Falls, chemical landfill

The frequency of isolation of three nonhomologous chlorobenzoate catabolic genotypes (clc, cba, and fcb) was determined for 464 isolates from freshwater sediments and groundwater in the vicinity of the Hyde Park industrial landfill site in the Niagara watershed. Samples were collected from both contaminated and noncontaminated sites during spring, summer, and fall and enriched at 4, 22, or 32 degrees C with micromolar to millimolar concentrations of chlorobenzoates and 3-chlorobiphenyl (M. C. Peel and R. C. Wyndham, Microb. Ecol: 33:59-68, 1997). Hybridization at moderate stringency to restriction-digested genomic DNA with DNA probes revealed the chlorocatechol 1,2-dioxygenase operon (clcABD), the 3-chlorobenzoate 3,4-(4,5)-dioxygenase operon (cbaABC), and the 4-chlorobenzoate dehalogenase (fcbB) gene in isolates enriched from all contaminated sites in the vicinity of the industrial landfill. Nevertheless, the known genes were found in less than 10% of the isolates from the contaminated sites, indicating a high level of genetic diversity in the microbial community. The known genotypes were not enriched from the noncontaminated control sites nearby. The clc, cba, and fcb isolates were distributed across five phenotypically distinct groups based on Biolog carbon source utilization, with the breadth of the host range decreasing in the order clc > cba > fcb. Restriction fragment length polymorphism (RFLP) patterns showed that the cba genes were conserved in all isolates whereas the clc and fcb genes exhibited variation in RFLP patterns. These observations are consistent with the recent spread of the cba genes by horizontal transfer as part of transposon Tn5271 in response to contaminant exposure at Hyde Park. Consistent with this hypothesis, IS1071, the flanking element in Tn5271, was found in all isolates that carried the cba genes. Interestingly, IS1071 was also found in a high proportion of isolates from Hyde Park carrying the clc and fcb genes, as well as in type strains carrying the clcABD operon and the biphenyl (bph) catabolic genes.     

Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.     

The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus

Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2–5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory system VraTSR, that is present in all S. aureus strains.     

Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene.

The Escherichia coli heat-stable enterotoxin STII gene in P307 is flanked by inverted repeat sequences, suggesting that the STII gene is part of a transposon. To study the transposability, a DNA fragment containing the putative STII transposon has been cloned. Results of transposition assays indicated that the STII gene can transpose from one plasmid to another. The size of the transposon has been determined to be approximately 9 kilobases. The structure and the location of the STII gene in clinical isolates of Escherichia coli have been investigated by restriction enzyme analyses. The structural genes of STII from different clinical isolates appear to be uniform in size, but the flanking sequences are heterogeneous. This result suggests that the STII genes in different isolates are not on the same transposon as observed in P307.     

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.     

Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene

Abstract: Fifty-five bacterial isolates, from English and French soils with different histories of carbofuran field treatment, which hydrolysed the N -methylcarbamate insecticide carbofuran to carbofuran 7-phenol were characterised phenotypically and genetically. The isolates were compared by using 125 physiological tests and morphological features, plasmid profiles and restriction fragment length polymorphism (RFLP) patterns of total DNA using the rRNA operon of Escherichia coli as a DNA probe. Cluster analysis of both phenotypic characters and RFLP patterns showed a high degree of diversity amongst the bacteria. Ten distinct plasmid profiles with 2–4 plasmids ranging in size from 84 to about 438 kb were visualised in 50 isolates. The majority of isolates had one of two types of plasmid profiles. Plasmid profiles and Eco RI restricted total DNA patterns were hybridised with an internal fragment of the carbofuran hydrolase ( mcd ) gene and 22 diverse soil isolates exhibited sequence homology with this gene probe. Our results indicate that sequences homologous to the mcd gene are located on a conserved Eco RI fragment (12 or 14 kb) of a plasmid (100, 105, 115 or 124 kb) found in diverse soil isolates from geographically distant areas. Thirty-three isolates did not exhibit detectable homology to the mcd gene probe and the hydrolase enzymes and genes in these isolates need further investigation.     

Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5' end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the "conserved" portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its "variable" portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this "conserved" cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.