Two distinct conjugal transfer systems on Streptomyces plasmid pZL1

The mechanism of conjugal transfer of plasmids in Gram-negative and unicellular Gram-positive bacteria is commonly via a type IV secretion system （T4SS） [1]. The genes encoding the T4S proteins are usually arranged in a single operon or a few operons. In Gram-negative Agrobacterium tumefaciens, the T4SS is encoded by the virB and virD operons （11 and 5 genes, respectively） [2]. Streptomyces are multicellular mycelial Gram-positive bac- teria that form unicellular spores. There are fundamental dif- ferences in the mechanisms of conjugal transfer between Streptomyces plasmids and those of Gram-negative and uni- cellular Gram-positive bacteria. Conjugal transfer of Streptomyces plasmids requires a tra gene encoding an FtsK/SpolIIE-family DNA translocator and a few adjacent genes [3]. No bacterial T4SS has previously been found on Streptomyces conjugative plasmids. We reported here the co- existence of both a T4SS-like and an FtsK/SpolIIE-family DNA translocator on a 128-kb Streptomyces plasmid, pZL 1.     

Functional organization of MobB, a small protein required for efficient conjugal transfer of plasmid R1162

MobB is a small (molecular weight = 15,097) protein encoded by the broad-host-range plasmid R1162 and is required for its efficient transfer by conjugation. The C-terminal half of the protein contains a membrane domain essential for transfer. This region can be replaced by a putative membrane domain from another, unrelated protein, and thus is likely to function independently from the rest of MobB. The other, functionally active region of MobB, identified by mutagenesis, is at the N-terminal end. One mutation affecting this region inhibits replication, suggesting that this part of the protein is contacting and sequestering the relaxase-linked primase. The overall organization reflects a multimeric and bipolar organization, with molecules of MobB anchored in the membrane at one end and engaging the relaxase at the other. This arrangement could increase the transfer frequency by raising the probability of contact between the relaxase and the membrane-embedded, coupling protein for type IV secretion.     

A thiostrepton-inducible expression vector for use in Streptomyces spp

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.     

Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.     

Development of a temperature-inducible expression system for Streptomyces spp.

PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using the promoterless gene for the thermostable Thermus flavus malate dehydrogenase as an indicator, an induction of enzyme activity of as much as was observed in a temperature shift from 28 to 37 degrees C. Temperature downshift reestablished repression of Ptra, making these promoter cassettes very attractive for the temporally regulated expression of cloned genes in Streptomyces spp.     

High-frequency conjugal transfer of a gonococcal penicillinase plasmid.

Gonococci containing a 24 X 10(6)-dalton conjugal plasmid were able to mobilize for transfer a smaller, non-self-transmissible penicillinase (Pcr) plasmid with high frequency under appropriate conditions. In some strains, over 10% of donor colony-forming units transferred the Pcr plasmid in a mating of less than 2 h, which suggests that the conjugal system was naturally derepressed. Colony-opacity variants containing different quantities of an approximately 28,000-dalton outer membrane protein were altered in their ability to act as conjugal donors and recipients. Maximal transfer of the Pcr plasmid was observed between transparent donors and recipients, lacking appreciable amounts of the 28,000-dalton protein. Under conditions of high-frequency Pcr plasmid mobilization, no conjugal mobilization of chromosomal markers could be discerned.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Melanin production as a visual indicator of conjugal transfer in Streptomyces

To visualize transfer of plasmid in Streptomyces during conjugation, we constructed a conjugative plasmid that harbored melC operon encoding an extracellular tyrosinase and placed it in...     

A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid

Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.     

Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

Isolation and preliminary characterization of a plasmid mutant derepressed for conjugal transfer in Staphylococcus aureus

The plasmid pCRG1600 is a 52.9-kb self-transmissible plasmid coding for resistance to aminoglycoside and β-lactam antibiotics in Staphylococcus aureus. When transferred by transduction, plasmid deletion mutants affecting one or more antibiotic-resistance genes were readily obtained. Of these, one derivative (pCRG1690) was found to exhibit a conjugal transfer frequency ca. 100-fold higher than that of the wild-type plasmid. A preliminary physical-genetic map of pCRG1600 located tra in a 14.6-kb region within the 16.9-kb XbaI-A fragment. An 8.5-kb deletion to the left of tra in pCRG1690 was specifically associated with the increased conjugal transferability of the plasmid. Thus, pCRG1690 appears similar to plasmids derepressed for conjugal transfer (drd) in gram-negative bacterial species.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Effect of chlorpromazine on conjugal plasmid transfer and sex pili

The major tranquillizer chlorpromazine (Cpz) inhibited the conjugal transfer of R and F'lac plasmids. The frequency of transfer of R-144 and R-100 plasmids was reduced with 2-3 log by Cpz at a concentration of 50-100 microgram/ml, while the frequency of RM-98 plasmid did not change under the same conditions. Cpz at 100 microgram/ml was an effective inhibitor of the transfer of F'lac plasmid. By means of electron microscopy and plaque assay, 100 microgram/ml Cpz was shown to reduce the adsorption rate of male specific ribonucleic acid phages MS-2 to the sides of F-pili. Common pili and flagellae seemed to be intact, but sex pili probably retracted in the presence of Cpz.     

Genetic organization and conjugal plasmid DNA transfer of pHP69, a plasmid from a Korean isolate of Helicobacter pylori

We isolated pHP69, a 9,153 bp plasmid from Helicobacter pylori with a 33.98% (G+C) content. We identified 11 open reading frames (ORFs), including replication initiation protein A (repA), fic (cAMP-induced filamentation protein), mccC, mccB, mobA, mobD, mobB, and mobC, as well as four 22 bp tandem repeat sequences. The nucleic acid and predicted amino acid sequences of these ORFs exhibited significant homology to those of other H. pylori plasmids. pHP69 repA encodes a replication initiation protein and its amino acid sequence is similar to those of replicase proteins from theta-type plasmids. pHP69 contains two types of repeat sequences (R1 and R2), a MOBHEN family mobilization region comprising mobC, mobA, mobB, and mobD, and genes encoding microcin B and C. Among the 36 H. pylori strains containing plasmids, mobA or mccBC are present in 12 or 6, respectively and 3 contain both genes. To examine intrinsic capability of H. pylori for conjugative plasmid transfer, a shuttle vector pBHP69KH containing pHP69 and replication origin of pBR322 was constructed. It was shown that this vector could stably replicate and be mobilized among clinical H. pylori strains and demonstrated to gene transfer by natural plasmid.     

Direct conjugal transfers of Ti plasmid to soil microflora

The bacterial species in soil that can receive a Ti plasmid by conjugation from Agrobacterium spp. were investigated. In order to have direct access to the potential reservoir of Ti plasmid amongst soil microflora, the conjugal system consisting of a multiply auxotrophic derivative of C58 (ST-96-4) and a derivative of pTiC58Delta(acc)R (pSTiEGK) containing a triple antibiotic-resistance cassette in traM was used to transfer the Ti plasmid in a complex soil microflora used as the recipient. Numerous transconjugants were obtained by this method but none was identified as Agrobacterium. This could be explained by the low density of Agrobacterium in the tested soil. As indicated by analysis of the ribosomal gene rrs, transconjugants recovered directly from soil were found to be new bacterial species which appeared to be closely related to Sinorhizobium spp.     

Expression of a Streptomyces plasmid promoter in Escherichia coli

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.     

Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F⁺) to a recipient (F⁻) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F⁺ transconjugants and the recovery time between successive conjugation events of the F⁺ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10⁻¹⁰ (cells/mL · min)⁻¹. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F⁺ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.