The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6

Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate. Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816. In contrast to the naphthalene-degrading pseudomonads, S. xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts. Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads. According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2′-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads. The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40–60% sequence identity to the isofunctional enzymes from the pseudomonads. In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied. S. xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes. Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models. Received 02 April 1999/ Accepted in revised form 09 July 1999     

The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs)

Many members of the sphingomonad genus isolated from different geological areas can degrade a wide variety of polycyclic aromatic hydrocarbons (PAHs) and related compounds. These sphingomonads such as Sphingobium yanoikuyae strain B1, Novosphingobium aromaticivorans strain F199, and Sphingobium sp. strain P2 have been found to possess a unique group of genes for aromatic degradation, which are distantly related with those in pseudomonads and other genera reported so far both in sequence homology and gene organization. Genes for aromatics degradation in these sphingomonads are complexly arranged; the genes necessary for one degradation pathway are scattered through several clusters. These aromatic catabolic gene clusters seem to be conserved among many other sphingomonads such as Sphingobium yanoikuyae strain Q1, Sphingomonas paucimobilis strain TNE12, S. paucimobilis strain EPA505, Sphingobium agrestis strain HV3, and Sphingomonas chungbukensis strain DJ77. Furthermore, some genes for naphthalenesulfonate degradation found in Sphingomonas xenophaga strain BN6 also share a high sequence homology with their homologues found in these sphingomonads. On the other hand, protocatechuic catabolic gene clusters found in fluorene-degrading Sphingomonas sp. strain LB126 appear to be more closely related with those previously found in lignin-degrading S. paucimobilis SYK-6 than the genes in this group of sphingomonads. This review summarizes the information on the distribution of these strains and relationships among their aromatic catabolic genes.     

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.     

Isolation and characterization of a novel sphingomonad capable of growth with chrysene as sole carbon and energy source

A bacterial strain able to grow in pure culture with chrysene as sole added carbon and energy source was isolated from PAH-contaminated soil after successive enrichment cultures in a biphasic growth medium. Initially, growth occurred in the form of a biofilm at the interface between the aqueous and non-aqueous liquid phases. However, after a certain time, a transition occurred in the enrichment cultures, with growth occurring in suspension and a concomitant increase in the rate of chrysene degradation. The strain responsible for chrysene degradation in these cultures, named Sphingomonas sp. CHY-1, was identified by 16S rDNA sequencing as a novel sphingomonad, the closest relative in the databases being Sphingomonas xenophaga BN6T (96% sequence identity). Both these strains clustered with members of the genera Sphingobium and Rhizomonas, but could not be categorically assigned to either genus. Sphingomonas sp. CHY-1 was characterized in terms of its growth on chrysene and other PAH, and the kinetics of chrysene degradation and 14C-chrysene mineralization were measured. At an initial chrysene concentration of 0.5 g l(-1) silicone oil, and an organic/aqueous phase ratio of 1:4, chrysene was 50% degraded after 5 days incubation and 97.5% degraded after 35 days. The protein content of cultures reached a maximum value of 11.5 microg ml(-1) aqueous phase, corresponding to 92 mg g(-1) chrysene. 14C-labelled chrysene was 50% mineralized after 6-8 weeks incubation, 10.7% of the radioactivity was incorporated into cell biomass and 8.4% was found in the aqueous culture supernatant. Sphingomonas sp. CHY-1 also grew on naphthalene, phenanthrene and anthracene, and naphthalene was the preferred substrate, with a doubling time of 6.9 h.     

Identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Antibiotic resistance study of clinical strains of Pseudomonas aeruginosa]

The study of 40 clinical strains of Ps. aeruginosa isolated from the wound surfaces of the patients showed that all the isolates were resistant to one or several antibiotics. The number of the strains resistant to 5, 4, 3, 2 or 1 drug was 5, 22.5, 25. 30 or 17.5 per cent respectively. Fifteen strains carried resistance plasmids capable of conjugative transfer. Eleven out of 21 plasmids controlled resistance to chloramphenicol, 7 plasmids controlled resistance to streptomycin and sulfanylamides, 1 plasmid controlled resistance to streptomycin and chloramphenicol. The presence of two types of the plasmids controlling resistance to chloramphenicol and streptomycin + sulfanylamides respectively was found. All the plasmids proved to be capable of conjugative transfer between the strains of Ps. aeruginosa ML (PAO). The frequency of the plasmid conjugative transfer in such crosses ranged from 10(-6) to 10(-3). Most of the plasmids belonged to the incompatibility groups P-2 and P-7. One plasmid belonged to the incompatibility group P-5. It should be noted that about a half of the plasmids (11 out of 21) belonged to the incompatibility group P-7 which up to the present time was conditional, since was represented by a single plasmid Rms 148.     

Evidence for the role of horizontal transfer in generating pVT1, a large mosaic conjugative plasmid from the clam pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600(T) reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.     

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.     

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli.

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.     

Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.     

Characterization of the Tra2 Region of the IncHI1 Plasmid R27

In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined. This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation. DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order. The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region. The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species. This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.