Application of intergeneric conjugation of <Emphasis Type="Italic">Escherichia coli — Streptomyces</Emphasis> for transfer of recombinant DNA into the strain <Emphasis Type="Italic">S. nogalater</Emphasis> IMET43360

Successful transfer of the plasmids studied here into S. nogalater IMET43360 cells by means of intergeneric conjugation in the system E. coli — Streptomyces has made the method a convenient means of constructing this strain. Through the use of DNA-DNA hybridization, the nature of the integration of the plasmids pVWB and pRT801 is determined and their influence on nogalamycin biosynthesis is studied. The results of our studies will help in gaining a more detailed understanding of the functioning of genes involved in the biosynthesis of nogalamicin in S. nogalater IMET43360. The use of conjugation for substitution and destruction of genes and heterologous expression makes it possible to obtain new “hybrid” antibiotics that can be produced by this strain.     

High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes

Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1. and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 10⁴-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with 'suicide' delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.     

An efficient gene transfer system for the pimaricin producer Streptomyces natalensis

Streptomyces natalensis produces the antifungal polyene macrolide pimaricin. Genetic manipulation of its biosynthetic genes has been hampered by the lack of efficient gene transfer systems. We have developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Using this approach, we managed to attain transformation efficiencies of 1 x 10(-4) exconjugants per recipient when using self-replicating vectors such as pHZ1358. The use of integrative vectors such as pSET152 or pSOK804 resulted in significantly lower efficiencies. Site-specific integration or the use of self-replicating plasmids did not affect pimaricin production or the essential functions of S. natalensis. Use of DNA methylation proficient E. coli donor strains resulted in no transformants, indicating the presence of methyl-specific restriction systems in S. natalensis. This methodology will enable easier manipulation of the genes responsible for pimaricin biosynthesis, and could prove valuable for the generation of new designer polyene macrolides with better antifungal activity and pharmacological properties. As an example of the validity of the method, we describe the introduction of Supercos-1-derived cosmid vectors into S. natalensis in order to promote gene replacements by double crossover recombination.     

Molecular characterization of plasmids pS7a and pS7b from Lactococcus lactis subsp. lactis bv. diacetylactis S50 as a base for the construction of mobilizable cloning vectors

Aims:  Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar.
Methods and Results:  Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four Eco RI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells.
Conclusions:  Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids.
Significance and Impact of the Study:  Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis .
Methods and Results:  The effect of DNA desalting, wall-weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis . By using this improved method, the greatest efficiency was reached 2 × 10¹⁰ CFU  μ g⁻¹ with pHT304, which is 10⁴ times higher than previously reported. Four large plasmids (29·1, 44·9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully.
Conclusions:  This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures.
Significance and Impact of the Study:  The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis . This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis . It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.     

Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPF_T-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPF_F nor MPF_I could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPF_T-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

Identification, cloning and sequencing of Escherichia coli strain χ1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran

Aims:  To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain χ1378 isolated from Iranian poultry and to predict its protein product, Iss.
Methods and Results:  The iss gene from E. coli strain χ1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain χ1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains.
Conclusions:  The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC).
Significance and Impact of the study:  Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.     

Transgenosis with participation of plasmid RP1; indications of the presence of a "composit plasmid" in an interspecies hybrid of Escherichia coli]

One of the transconjugants (1-7) obtained by the authors earlier in the conjugation of Escherichia coli J-62 with Pseudomonas aeruginosa 1822, besides the plasmic RP1 has acquired the ability to grow without proline and tryptophan. The detailed analysis has shown that in the conjugation of the transconjugant 1-7 with different strains of E. coli the plasmic RP1 and chromosomal genes were transmitted together, but in transduction--by means of bacteriophage P1, independently of each other. The fertility was found only in the transductants carrying the plasmid RP1. This suggests that in the intergeneric conjugations the transmission of chromosomal genes may occur without any firm link with the plasmid (as in the case of "aggregated plasmids"). In E. coli cells these chromosomal fragments of Ps. aeruginosa apparently formed small nontransmissible replicons.     

The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans.

Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.     

A Non-Restricting and Non-Methylating <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain for DNA Cloning and High-Throughput Conjugation to <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.     

Vancomycin-resistant Enterococcus spp. in marine environments from the West Coast of the USA

Aims:  The aim of the study was to determine if vancomycin-resistant Enterococcus spp. [VRE] carrying vanA and/or vanB genes were present in public marine beaches and a fishing pier [2001–2003, 2008] from Washington and California [2008].
Methods:  PCR assays for the vanA and/or vanB genes with verification by DNA–DNA hybridization of the PCR products were used. Positive isolates were speciated using the BD BBL Crystal™ Identification and/or by sequencing the 16S ribosomal region.
Results:  Eighteen (8%) of 227 isolates including Enterococcus faecalis , Enterococcus faecium , Enterococcus casseliflavus/gallinarum and a Staphylococcus epidermidis carrying vanA and/or vanB genes, from four of six Washington and one of two California sites, were identified. Selected VRE and the S. epidermidis were able to transfer their van genes to an E. faecalis recipient at frequencies ranging from 1·9 × 10⁻⁶ to 6·7 × 10⁻⁹.
Conclusions:  Vancomycin-resistant Enterococcus spp. was isolated from five of the seven sites suggesting that other North America public beaches could be the reservoirs for VRE and should be assessed.
Significance & Impact of the Study:  This is the first report of isolation and characterization of VRE strains (and a vanB Staphylococcus sp.) from North American environmental sources suggesting that public beaches may be a reservoir for possible transmission of VRE to beach visitors.     

Fate of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry applied to soil surrounding tall fescue

Aim:  To investigate the potential transfer of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry into the tissue of tall fescue plants.
Methods and Results:  Tall fescue plants ( n  =   50) were fertilized with a manure slurry inoculated with E. coli O157:H7 and Salmonella . Soil was collected and tall fescue plants ( n  =   10 per day) harvested on day 1, 2, 4, 8, and 14 after manure slurry fertilization. Soil samples were positive for E. coli O157:H7 on all days and on day 1, 2, 8, and 14 for Salmonella . None of the plant tissue samples were positive for E. coli O157:H7 on day 1 or 2; however, 20%, 30% and 40% of plant tissue samples were positive for E. coli O157:H7 on day 4, 8, and 14, respectively.
Conclusions:  It may be possible that E. coli O157:H7 can become transmitted and internalized into tall fescue plant tissue within 4 days after exposure to an E. coli O157:H7-contaminated manure slurry. Salmonella did not appear to be transferred to tall fescue plant tissue.
Significance and Impact of the Study:  Faeces contaminated with E. coli O157:11H7 may be one means by which grazing ruminants spread bacterial pathogens to additional animals.     

Development of a system of plasmid transfer in the strains Streptomyces avermitilis ATCC 31272 and Saccharopolyspora erythraea ATCC 11635 by the method of intergeneric conjugation

A new method of plasmid DNA transfer from the donor strain Escherichia coli S17-1 to the erythomycin-producing strain Saccharopolyspora erythraea and avermectin-producing strain Streptomyces avermitilis via intergeneric conjugation was proposed. The optimal parameters of the method were chosen for increasing the efficiency of crosses and ensuring easily reproducible results. The behavior of the multicopy plasmid pPM803 and the integration vector pTO1 along with a number of new plasmids specially created by us, was examined in these strains. A new plasmid vector (pSI60) capable of integrating into the chromosome of actinomycetes at the integration site of the temperate actinophage phi C31 was constructed. This vector possesses unique sites convenient for cloning and may be stably maintained in exconjugants of S. avermitilis and in the model strain Streptomyces lividans. The gene encoding resistance to spectinomycin and streptomycin was cloned into the vector pSI60 in this strain. For cloning in strain Sac. erythraea, vectors pSI261-280, which integrate into the chromosome via homology with the cloned DNA and can be stably maintained in exconjugants, were constructed.     

Plasmid transfer to Bordetella pertussis: conjugation and transformation

Plasmids of the P and W incompatibility groups were introduced into Bordetella pertussis by conjugation. Plasmid DNA isolated from B. pertussis could be reintroduced by transformation. DNA isolated from Escherichia coli could not be introduced into B. pertussis by transformation if this DNA contained HindIII restriction sites. We have demonstrated that HindIII sites are modified by B. pertussis. Plasmids of the FI and FII incompatibility groups could not be introduced into B. pertussis by conjugation, and nonconjugative plasmids of the ColE1 and Q incompatibility groups could not be introduced by transformation. Our ability to introduce plasmids in the laboratory suggests that the apparent lack of plasmids in natural isolates of B. pertussis is not due to an inability to act as a plasmid recipient.     

Screening for yeast mutants defective in recipient ability for transkingdom conjugation with Escherichia coli revealed importance of vacuolar ATPase activity in the horizontal DNA transfer phenomenon

Proteobacterium Escherichia coli strains harboring wide-transfer-range conjugative plasmids are able to transfer these plasmids to several yeast species. Whole plasmid DNA is mobilizable in the transkingdom conjugation phenomenon. Owing to the availability of various conjugative plasmids in bacteria, the horizontal DNA transfer has potential to occur between various bacteria and eukaryotes. In order to know host factor genes involved in such conjugation, we systematically tested the conjugability of strains among a yeast library comprising single-gene-knockout mutants in this study. This genome-wide screen identified 26 host chromosomal genes whose absence reduced the efficiency of the transkingdom conjugation. Among the 26 genes, 20 are responsible for vacuolar ATPase activity, while 5 genes (SHP1, CSG2, CCR4, NOT5, and HOF1) seem to play a role in maintaining the cell surface. Lack of either ZUO1 gene or SSZ1 gene, each of which encodes a component of the ribosome-associated cytoplasmic molecular chaperone, also strongly affected transkingdom conjugation.