Gene replacement in mycobacteria by using incompatible plasmids

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.     

The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors

Escherichia coli-mycobacteria shuttle vectors, derived from pAL5000 (a mycobacterial plasmid) and pUC19, were frequently found to undergo structural alterations due to transposition of IS1096, a Mycobacterium smegmatis transposable element, at a cluster of sites located within a small region of 60 bp, immediately upstream of a kanamycin resistance gene present in these vectors. The structural alterations led to deletion of large regions of the vector which, in several cases, were found to extend into the ORF2 (RepB) coding sequences of the pAL5000 replication region without affecting its replication capability. This suggests that the entire ORF2 coding sequences of the pAL5000 replication region may not be essential for replication of pAL5000-derived vectors. The deletion derivatives, which contain the minimal sequences required for replication and selection in mycobacteria, were found to be structurally stable and therefore these could be potentially used as stable vector systems for the transformation of mycobacteria.     

Versatile <Emphasis Type="Italic">Rhodococcus equi</Emphasis>–<Emphasis Type="Italic">Escherichia coli</Emphasis> shuttle vectors

Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.     

The genetics ofMycobacterium tuberculosis

Gene manipulation inMycobacterium tuberculosis has been slow in coming of age owing to the inherent difficulties associated with working on this aerosol-transmitted pathogen, in addition to the paucity of molecular tools such as plasmids and transposons. One of the early approaches to overcome these difficulties was the development of phasmids, which combined the properties of phages and plasmids and allowed introduction of recombinant genes into mycobacteria. The lone plasmid pAL5000 of mycobacteria has been exploited to its fullest potential in the construction of a plethora of vectors. Above all, the single most important achievement has been the development of elegant and innovative approaches to overcome the problem of illegitimate recombination which threatened the success of allelic-exchange mutagenesis in the slow-growing pathogenic mycobacterial species. In this review I discuss the current status of conditionally replicating plasmid and transposon vectors and their application in generating targeted mutations in mycobacteria.     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids.     

Use of the pAL5000 replicon in PAH-degrading mycobacteria: application for strain labelling and promoter probing

Three environmental Mycobacterium strains (LB501T, LB307T and VM552) able to degrade anthracene, phenanthrene or pyrene, respectively, were successfully electroporated with pAL5000-based plasmids containing the green fluorescent protein (gfp) gene of Aequoria victoria under the control of the hsp60 promoter of Mycobacterium bovis following a slightly modified standard procedure. Transformants showed irregular gfp expression profiles. Four plasmid derivatives were constructed that contained gene promoters isolated from, and adapted to, gene expression in polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria. One derivative directed strong and homogeneous expression of GFP, allowing dual analysis of both GFP- and PAH-derived fluorescence as assessed by confocal laser scanning microscopy. The results reported here demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNA-based studies in PAH-degrading Mycobacterium spp.     

DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in gram-positive Actinomycetes bacteria. The replication regions of these plasmids comprise a bicistronic operon, repA-repB, encoding two replication proteins. Located upstream is a cis-acting element that functions as the origin of replication. It comprises an ~200-bp segment spanning two binding sites for the replication protein RepB, a low-affinity (L) site and a high-affinity (H) site separated by an ~40-bp spacer sequence. The trajectory of the DNA in the RepB-origin complex has been investigated, and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long-range polar curvature which affected the DNA sequences 3′ to the H site. These movements appear to be essential for the in-phase alignment of the L and H sites that leads to the formation of a looped structure. A novel property of RepB unearthed in this study is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origins of replication of pAL5000 and related plasmids are highly flexible and that multimeric, RepB-like initiator proteins bind the origin and induce local deformations and long-range curvatures which are probably necessary for the proper functioning of the origin.     

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids. Received: 13 November 1997 / Accepted: 17 February 1998     

Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics.     

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and γ irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.     

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.     

An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose

Counterselectable markers are powerful tools in genetics because they allow selection for loss of a genetic marker rather than its presence. In mycobacteria, a widely used counterselectable marker is the gene encoding levan sucrase (sacB), which confers sensitivity to sucrose, but frequent spontaneous inactivation complicates its use. Here we show that the Escherichia coli galactokinase gene (galK) can be used as a counterselectable marker in both Mycobacterium smegmatis and Mycobacterium tuberculosis. Expression of E. coli galK, but not the putative M. tuberculosis galK, conferred sensitivity to 2-deoxy-galactose (2-DOG) in both M. smegmatis and M. tuberculosis. We tested the utility of E. coli galK as a counterselectable marker in mycobacterial recombination, both alone and in combination with sacB. We found that 0.5% 2-DOG effectively selected recombinants that had lost the galK marker with the ratio of galK loss/galK mutational inactivation of approximately 1:4. When we combined galK and sacB as dual counterselectable markers and selected for dual marker loss on 0.2% 2-DOG/5% sucrose, 98.6–100% of sucrose/2-DOG resistant clones had undergone recombination, indicating that the frequency of mutational inactivation of both markers was lower than the recombination frequency. These results establish a new counterselectable marker system for use in mycobacteria that can shorten the time to generate unmarked mutations in M. smegmatis and M. tuberculosis.     

Relationships between autonomous and integrated forms of tetracycline resistance plasmid in Staphylococcus aureus.

Recombinant plasmids carrying apparently the complete genome of a small staphylococcal plasmid, pT181, or of its temperature-sensitive replication mutant, pSA0301, were isolated and characterized; in these recombinants, pT181 or pSA0301 were considered as “integrated” into the other plasmid, inasmuch as they seem to have a subsidiary role in the replication of the respective recombinant plasmids. Using these recombinants, the incompatibility relationships between integrated and autonomous forms of the same plasmid were studied. The results obtained showed that, although integrated plasmids express their incompatibility toward autonomous ones, they are not susceptible to the incompatibility manifested by an autonomous or another integrated plasmid. No differences were observed between pT181 and pSA0301 in their response to the incompatibility manifested by recombinant plasmids. The expression of the incompatibility of an integrated plasmid did not require the function of the repC gene, involved in plasmid autonomous replication. Moreover, the pT181 repC⁺ gene seems not to be expressed when pT181 is integrated into another plasmid in that the integrated form does not complement autonomous pSA0301 for replication at nonpermissive temperature.     

Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors

Summary A high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha.     

Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility

The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.     

The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110

Gateway^® cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method.