Broad Host Range Conjugative Plasmid Research Articles

Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.

Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Targeting antimicrobial resistance genes in clinical isolates from healthcare-associated infections using CRISPR-Cas9

Of the numerous approaches to counteracting the spread of antimicrobial resistance (AMR), one that is receiving increased attention is the use of CRISPR-Cas-based gene editing technology to remove resistance-conferring sequences from bacteria. Proof-of-principle studies have shown that CRISPR-Cas is able to successfully remove AMR genes from monocultures or very simple bacterial communities in the laboratory, but these constructs and their delivery must be adapted to be used in real-world medical settings. One area where such a technology may be implemented is tackling healthcare-associated infections. Development of a CRISPR-Cas9 cassette that is able to target particular resistance genes would be a powerful tool in addressing these infections. CRISPR-Cas9 will therefore be implemented to target AMR genes in clinical isolates of vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase (CTX-M) or carbapenemase (Oxa-48)-producing Klebsiella pneumoniae and Escherichia coli, by utilising a broad host range conjugative plasmid to deliver this construct via conjugation. Preliminary work using E. coli MG1655 as plasmid donor have shown that all strains can receive the plasmid, and there is significant variation in conjugation frequency between E. coli recipients.

AMR gene removal by conjugative delivery of CRISPR-Cas9

Antimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed.

Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System.

Antibacterial resistance necessitates the development of novel treatment methods for infections. Protein aggregates have recently been applied as antimicrobials to disrupt bacterial homeostasis. Past work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding a de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decrease. Further, we developed a probiotic delivery system using the broad-host range conjugative plasmid RK2 to transfer the gene from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying opaL by adding the RK2 origin of transfer. We show that opaL is nontoxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase-expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control T7 RNA polymerase expression and provides a basis for the design and delivery of aggregating antimicrobial peptides.

Plasmids can transfer to Clostridium difficile CD37 and 630Δerm both by a DNase resistant conjugation-like mechanism and a DNase sensitive mechanism.

Broad host range conjugative plasmids that replicate in Escherichia coli have been widely used to mobilise smaller replicons, bearing their cognate origin of transfer (oriT) into a variety of organisms that are less tractable genetically, such as Clostridium (Clostridioides) difficile. In this work we demonstrated that the oriT region of pMTL9301 (derived from RK2) is not required for transfer between E. coli and C. difficile strains 630Δerm and CD37 and that this oriT-independent transfer is abolished in the presence of DNase when CD37 is the recipient. Transfer to the 630Δerm strain is DNase resistant even without an obvious oriT, when E. coli CA434 is used as a donor and is sensitive to DNase when E. coli HB101 is the donor.

Identification of oriT and a recombination hot spot in the IncA/C plasmid backbone

Dissemination of multiresistance has been accelerating among pathogenic bacteria in recent decades. The broad host-range conjugative plasmids of the IncA/C family are effective vehicles of resistance determinants in Gram-negative bacteria. Although more than 150 family members have been sequenced to date, their conjugation system and other functions encoded by the conserved plasmid backbone have been poorly characterized. The key cis-acting locus, the origin of transfer (oriT), has not yet been unambiguously identified. We present evidence that IncA/C plasmids have a single oriT locus immediately upstream of the mobI gene encoding an indispensable transfer factor. The fully active oriT spans ca. 150-bp AT-rich region overlapping the promoters of mobI and contains multiple inverted and direct repeats. Within this region, the core domain of oriT with reduced but detectable transfer activity was confined to a 70-bp segment containing two inverted repeats and one copy of a 14-bp direct repeat. In addition to oriT, a second locus consisting of a 14-bp imperfect inverted repeat was also identified, which mimicked the function of oriT but which was found to be a recombination site. Recombination between two identical copies of these sites is RecA-independent, requires a plasmid-encoded recombinase and resembles the functioning of dimer-resolution systems.

Whole Genome Sequence Analysis of the First Australian OXA-48-Producing Outbreak-Associated Klebsiella pneumoniae Isolates: The Resistome and In Vivo Evolution

Whole genome sequencing was used to characterize the resistome of intensive care unit (ICU) outbreak-associated carbapenem-resistant K. pneumoniae isolates. Importantly, and of particular concern, the carbapenem-hydrolyzing β-lactamase gene bla OXA-48 and the extended-spectrum β-lactamase gene bla CTX-M-14, were identified on a single broad host-range conjugative plasmid. This represents the first report of bla OXA-48 in Australia and highlights the importance of resistance gene surveillance, as such plasmids can silently spread amongst enterobacterial populations and have the potential to drastically limit treatment options. Furthermore, the in vivo evolution of these isolates was also examined after 18 months of intra-abdominal carriage in a patient that transited through the ICU during the outbreak period. Reflecting the clonality of K. pneumoniae, only 11 single nucleotide polymorphisms (SNPs) were accumulated during this time-period and many of these were associated with genes involved in tolerance/resistance to antibiotics, metals or organic solvents, and transcriptional regulation. Collectively, these SNPs are likely to be associated with changes in virulence (at least to some extent) that have refined the in vivo colonization capacity of the original outbreak isolate.

Structure-Function Analysis of Escherichia coli DNA Helicase I Reveals Non-overlapping Transesterase and Helicase Domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.

Characterisation and genetic organisation of a 24-MDa plasmid from the Brazilian Purpuric Fever clone of Haemophilus influenzae biogroup aegyptius

Strains of Haemophilus influenzae biogroup aegyptius causing septicaemia were identified in Brazil in the 1980s, causing the life-threatening illness of Brazilian Purpuric Fever (BPF). The strains were found to fall into a single clonal group, the BPF clone, characterised by their possession of the ∼24 MDa “3031” plasmid. In this work we report the characterisation and genetic organisation of this plasmid. Analysis of the gene content of what appears to be a typical broad host range conjugative plasmid, its presence in non-BPF strains as revealed by Southern hybridisation, and the recent discovery of plasmid-lacking BPF strains, has led us to conclude that it is unlikely to play a critical role in bacterial virulence. Establishing its entire sequence has nonetheless been an important step on the road to delineating, by comparison of BPF and non-BPF strains, chromosomal genetic loci that are involved in the special virulence of the BPF clone.

Dissemination of catabolic plasmids among desiccation-tolerant bacteria in soil microcosms.

The dissemination of catabolic plasmids was compared to bioaugmentation by strain inoculation in microcosm experiments. When Rhodococcus erythropolis strain T902, bearing a plasmid with trichloroethene and isopropylbenzene degradation pathways, was used as the inoculum, no transconjugant was isolated but the strain remained in the soil. This plasmid had a narrow host range. Pseudomonas putida strain C8S3 was used as the inoculum in a second approach. It bore a broad host range conjugative plasmid harboring a natural transposon, RP4::Tn4371, responsible for biphenyl and 4-chlorobiphenyl degradation pathways. The inoculating population slowly decreased from its original level (10(6) colony-forming units [CFU]/g of dry soil) to approx 3 x 10(2) CFU/g of dry soil after 3 wk. Transconjugant populations degrading biphenyl appeared in constant humidity soil (up to 2 x 10(3) CFU/g) and desiccating soil (up to 10(4) CFU/g). The feasibility of plasmid dissemination as a bioaugmentation technique was demonstrated in desiccating soils. The ecologic significance of desiccation in bioaugmentation was demonstrated: it upset the microbial ecology and the development of transconjugants.

A TEM-2beta-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia.

A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM beta-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.

Recombinant plasmid DNA mobilization by activated sludge strains grown in fixed-bed or sequenced-batch reactors

The possibilities of mobilizing recombinant DNA (rDNA) by strains from activated sludge have been studied. A mixture of ten bacterial strains isolated from activated sludge was grown on a support in a continuously fed fixed-bed reactor or in suspension in a sequenced-batch reactor with biomass recycling. After reaching steady state, the bacteria in both types of reactors were used as the recipient population for evaluating the dissemination of the non-conjugative and non-mobilizable recombinant plasmid pCE328. The dissemination of this plasmid, derived from pBR328 (tra−, mob−, oriT−), was studied in comparison with that of pCE325 (pBR325 derivative tra−, mob−, oriT+), and that of the natural broad host range conjugative plasmid R388 (IncW). Initially the mobilization properties of pBR type recombinant plasmids between two strains of Escherichia coli were determined in the fixed-bed reactor. Only the mobilization of the recombinant plasmid bearing a transfer origin (pCE325) was observed. The survival of E. coli strains bearing either the non-transferable plasmid pCE328 or the conjugative plasmid R388 and added to the activated sludge reactors was measured in the effluent of both types of reactors. In the fixed-bed reactor effluent, the donor populations remained stable after three days of operation and reached 103 to 105 colony forming units (cfu) ml−1, whereas they decreased in the sequenced-batch reactors: donors bearing the pCE328 plasmid decreased by 2 log units in 20 days, whereas those bearing the R388 plasmid decreased by less than 1 log unit. Mobilization of the pCE328 plasmid could not be detected in any case. Transfer of the conjugative plasmid R388, used as a positive propagation control, was detected in strains arising from activated sludge in both types of reactors. Nevertheless, establishment of transconjugants was observed only in the fixed biomass at the rate of 10−7 transconjugants per recipient.

Recombinant plasmid DNA mobilization by activated sludge strains grown in fixed-bed or sequenced-batch reactors

The possibilities of mobilizing recombinant DNA (rDNA) by strains from activated sludge have been studied. A mixture of ten bacterial strains isolated from activated sludge was grown on a support in a continuously fed fixed-bed reactor or in suspension in a sequenced-batch reactor with biomass recycling. After reaching steady state, the bacteria in both types of reactors were used as the recipient population for evaluating the dissemination of the non-conjugative and non-mobilizable recombinant plasmid pCE328. The dissemination of this plasmid, derived from pBR328 ( tra − , mob − , oriT − ), was studied in comparison with that of pCE325 (pBR325 derivative tra − , mob − , oriT + ), and that of the natural broad host range conjugative plasmid R388 (IncW). Initially the mobilization properties of pBR type recombinant plasmids between two strains of Escherichia coli were determined in the fixed-bed reactor. Only the mobilization of the recombinant plasmid bearing a transfer origin (pCE325) was observed. The survival of E. coli strains bearing either the non-transferable plasmid pCE328 or the conjugative plasmid R388 and added to the activated sludge reactors was measured in the effluent of both types of reactors. In the fixed-bed reactor effluent, the donor populations remained stable after three days of operation and reached 10 3 to 10 5 colony forming units (cfu) ml −1, whereas they decreased in the sequenced-batch reactors: donors bearing the pCE328 plasmid decreased by 2 log units in 20 days, whereas those bearing the R388 plasmid decreased by less than 1 log unit. Mobilization of the pCE328 plasmid could not be detected in any case. Transfer of the conjugative plasmid R388, used as a positive propagation control, was detected in strains arising from activated sludge in both types of reactors. Nevertheless, establishment of transconjugants was observed only in the fixed biomass at the rate of 10 −7 transconjugants per recipient.

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Transfer of the broad host range conjugative plasmids pAM β 1, pIP501 and its derivative pIL205 into Lactobacillus sake was studied. Frequencies up to 5 × 10 −6 transconjugants per recipient cell were found from Lactococcus lactis strain IL1403 to various strains of L. sake. The transfer takes place by a conjugation-like process on solid surface agar. Intrageneric transfer between L. sake strains was also observed, although at lower frequencies (10 −7 transconjugants per recipient cell). The ability to transfer these plasmids into L. sake strains which are either refractory to transformation or poorly transformable provides a method of genetic transfer, thus opening the way for cloning strategies developed in other Gram-positive bacteria.

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 inLactobacillus sake

Transfer of the broad host range conjugative plasmids pAM β 1, pIP501 and its derivative pIL205 into Lactobacillus sake was studied. Frequencies up to 5 × 10−6 transconjugants per recipient cell were found from Lactococcus lactis strain IL1403 to various strains of L. sake. The transfer takes place by a conjugation-like process on solid surface agar. Intrageneric transfer between L. sake strains was also observed, although at lower frequencies (10−7 transconjugants per recipient cell). The ability to transfer these plasmids into L. sake strains which are either refractory to transformation or poorly transformable provides a method of genetic transfer, thus opening the way for cloning strategies developed in other Gram-positive bacteria.

Plasmid Transfer by Electroporation and Conjugation in Tetragenococcus and Pediococcus Genera and Evidence of Plasmid-Linked Metabolic Traits

Various strains of Pediococcus genus were successfully transformed by electroporation using the broad host-range plasmid pSA3 and the lactococcal Rep22 based-replicon pUCB304. Failure to transform Tetragenococcus strains by electroporation have led to use conjugation as an alternative plasmid DNA transfer mechanism. Intergeneric matings conducted with the broad host-range conjugative plasmid pVA797 from Lactococcus lactis subsp. lactis SL2/797A to Tetragenococcus halophilus and Pediococcus pentosaceus strains have shown that Tetragenococcus strains are not impervious to plasmid DNA transfer. Plasmid and metabolic profiles of wild and mutant strains of Pediococcus pentosaceus NCDO990 have shown that many metabolic traits including lactose utilization are plasmid linked.

High Frequency Intergeneric and Intrageneric Conjugal Transfer of Drug Resistance Plasmids in Leuconostoc mesenteroides ssp. cremoris

Transfer of the broad host range conjugative plasmids pAMβ1, pVA797:: Tn917, and pIP501 between Lactococcus lactis and Leuconostoc mesenteroides ssp. cremoris occurred at high frequency via a conjugation-like process on solid surface agar. Frequencies of 10–1 to 10–3 transconjugants per input donor cell were observed from L. lactis to three strains of Leuc. mesenteroides ssp. cremoris. Reverse intergeneric transfer from Leuc. mesenteroides ssp. cremoris to L. lactis occurred at frequencies up to 10–4 transconjugants per donor cell. Intrageneric transfer between Leuc. mesenteroides ssp. cremoris strains was also observed at frequencies as high as 10–3 transconjugants per donor cell. The ability to transfer these plasmids to and among Leuc. mesenteroides ssp. cremoris at high frequencies provides a method of genetic transfer, making them available to cloning strategies developed in other Gram-positive bacteria.

A transposon vector for broad host range cloning

The genetic analysis of many procaryotes is hindered by the lack of suitable broad host range cloning vectors. These vectors are often relatively large with limited cloning possibilities and frequently exhibit instability in recipient cells (1). Furthermore, the antibiotic resistance markers are not always well expressed in the recipient. We have constructed a 6.9 kb vector, based on transposon Tn5, which allows transposition of cloned DNA onto any broad host range conjugative (BHRC) plasmid and subsequent gene transfer. This flexible approach alleviates the aforementioned problems. Plasmid pJP5011 consists of pJP5010 carrying transposon Tn5-lacZ' in the ampicillin resistance (Amr/) gene. Plasmid pJP5010 is pUC19 (2) with the lacZ' fragment deleted. Tn5-lacZ' is Tn5 carrying the lacZ' fragment of pUC19. HaeU was used for deletion and insertion of the lacZ' fragment. Plasmid pJP5011 has unique Sail, Accl, Xbal, BamHl, Xmal, Smal, Kpnl, Sacl and EcoRI restriction sites available for a-complementation cloning. This was confirmed by performing clonings without insert DNA and noting the absence of white colonies on selective MacConkey (Oxoid) or X-gal media. Using pJP5011, a number of DNA fragments have been transferred to Rhodobacter sphaeroides giving the expected resultant phenotype. Routinely, a BHRC plasmid is mated into the recombinant cells followed by mating with the recipient. Growth on an appropriate contraselective antibiotic and kanamycin indicates recombinant transfer via transposition. In some cases, kanamycin resistance is not well expressed in the recipient or the recipient mutates frequently to kanamycin resistance. In such situations, transposition can first be selected by transfer to another E. coli strain followed by the use of a BHRC plasmid marker to detect BHRC plasmid: :Tn5-insert transfer to the recipient. Selection of transposition in E. coli is also advisable in situations where conjugal transfer frequency to the recipient is low due to restriction or other factors. Some advantages of this system are: a) The selection marker employed can be varied with the use of alternate BHRC plasmids. b) BHRC plasmid: :Tn-5 insert DNA constructs are very stable in a wide variety of organisms. For example, the 28 kb carotenoid gene cluster, cloned in Tn5 and expressed in a number of gram negative organisms (3), has almost never shown loss or alteration of colour due to DNA rearrangement. This experience has been repeated with other cloned pathways (unpublished data), c) Inserts in Tn5 as large as 40 kb can be transposed to BHRC plasmids (3). d) The kanamycin resistance (KanO gene of Tn5 is well expressed in many organisms (4). e) The carrier replicon (BHRC, mobilizable, suicidal, viral etc.) can be varied according to need. For instance, stable chromosomal insertions of recombinant DNA could be obtained in recipients by using mobilizable suicide vectors. For transformable recipients, pJP5011 itself could be B-y~t J|!D

Heterospecific transcription of the Escherichia coli rpoB-3 allele in gram-negative bacteria.

SummaryrpoB is the structural gene for the β-subunit ofE. coliRNA polymerase. TherpoB-3 allele confers resistance to the antibiotic rifampicin and is unusual in being dominant to the wild-type allele. We used the plasmid pZD23, a derivative of the broad host range conjugative plasmid RP4, to introduce therpoB-3 allele into a range of bacterial species. Species belonging to the same family asE. coli(Enterobacter aerogenes, Citrobacter freundii, Hafnia alvei møller, Klebsiella pneumoniae, Salmonella typhimurium) expressedrpoB-3 to give a rifampicin resistant phenotype; this demonstrated heterospecific transcription. The transfer of pZD23 to the non-Enterobacteriaceae speciesAzotobacter vinelandiiandRhizobium leguminosarumdid not result in rifampicin resistance. In the former case this was due to non-expression of therpoB-3 resistance phenotype, in the latter case the dominant resistance phenotype had been lost from pZD23. Heterospecific transcription can be used as a criterion for the investigation of genetic relatedness between bacterial species.

Conjugation-mediated genetic exchange in Legionella pneumophila.

Genetic exchange mechanisms, to our knowledge, have not been reported for Legionella pneumophila, and consequently, studies on the genetic organization of L. pneumophila have not appeared in the literature. Here, we describe gene transfer mediated by broad host range conjugative plasmids in Legionella spp. Escherichia coli strains carrying plasmids RP1 and R68.45 (IncP1), S-a (IncW), and R40a (IncC), but not plasmids of incompatibility groups FI, FII, and FV, served as donors in matings with L. pneumophila Knoxville 1 (LPK-1). Transconjugants selected by resistance to kanamycin (RP1, R68.45, and S-a) and carbenicillin (R40a) were observed at frequencies of 6.6 X 10(-3), 4.7 X 10(-3), 2.2 X 10(-4), and 5.4 X 10(-5), respectively. Plasmid transfer was not affected by DNase added to the mating medium. After plasmid transfer, LPK-1 stably maintained RP1, R68.45, and S-a, but not R40a. Plasmid-containing LPK-1 isolates also served as donors in agar plate matings with E. coli W1485-1 and naladixic acid-resistant mutants of LPK-1, Legionella micdadei, and Legionella longbeachii. Recombinational exchange of a chromosomal trait was demonstrated when a thymidine auxotroph of L. pneumophila was repaired by R68.45-mediated chromosomal mobilization of a prototrophic donor strain.