Chromosomal DNA transfer in Mycobacterium smegmatis is mechanistically different from classical Hfr chromosomal DNA transfer

Classical conjugal DNA transfer of chromosomal DNA in bacteria requires the presence of a cis-acting site, oriT, in the chromosome. Acquisition of an oriT occurs if a conjugative plasmid integrates into the chromosome to form an Hfr donor strain, which can transfer extensive regions of chromosomal DNA. Because oriT sequences are unique, and because transfer occurs in a 5' to 3' direction, the frequency with which a particular gene is inherited by the recipient depends on the gene's location: those closest to the 3' side of oriT are transferred most efficiently. In addition, as the entire chromosome must be transferred to regenerate oriT, Hfr transconjugants never become donors. Here we describe novel aspects of a chromosomal DNA transfer system in Mycobacterium smegmatis. We demonstrate that there are multiple transfer initiations from a donor chromosome and, as a result, the inheritance of any gene is location-independent. Transfer is not contiguous; instead, multiple non-linked segments of DNA can be inherited in a recipient. However, we show that, with appropriate selection, segments of DNA at least 266 kb in length can be transferred. In further contrast to Hfr transfer, transconjugants can become donors, suggesting that the recipient chromosome contains multiple cis-acting sequences required for transfer, but lacks the trans-acting transfer functions. We exploit these observations to map a donor-determining locus in the M. smegmatis chromosome using genetic linkage analysis. Together, these studies further underline the unique nature of the M. smegmatis chromosomal transfer system.     

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae . The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis , which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc²155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli .     

The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis

Conjugal DNA transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmid-mediated DNA transfer. Previously, we had shown that the secretory apparatus, ESX-1, negatively regulated DNA transfer from the donor strain; ESX-1 donor mutants are hyper-conjugative. Here, we describe a genome-wide transposon mutagenesis screen to isolate recipient mutants. Surprisingly, we find that a majority of insertions map within the esx-1 locus, which encodes the secretory apparatus. Thus, in contrast to its role in donor function, ESX-1 is essential for recipient function; recipient ESX-1 mutants are hypo-conjugative. In addition to esx-1 genes, our screen identifies novel non- esx-1 loci in the M. smegmatis genome that are required for both DNA transfer and ESX-1 activity. DNA transfer therefore provides a simple molecular genetic assay to characterize ESX-1, which, in Mycobacterium tuberculosis , is necessary for full virulence. These findings reinforce the functional intertwining of DNA transfer and ESX-1 secretion, first described in the M. smegmatis donor. Moreover, our observation that ESX-1 has such diametrically opposed effects on transfer in the donor and recipient, forces us to consider how proteins secreted by the ESX-1 apparatus can function so as to modulate two seemingly disparate processes, M. smegmatis DNA transfer and M. tuberculosis virulence.     

Retrotransfer of DNA in the rhizosphere

Retrotransfer of DNA refers to the phenomenon by which a plasmid travels from a host strain to a recipient one and returns to the original host, bringing with it DNA from the recipient. The resultant host strain with DNA from the recipient is called a retrotransconjugant. The retrotransfer phenomenon mediated by the TOL plasmid pWW0 and other plasmids has been documented on plates under optimal laboratory culture conditions, but never under natural conditions. In this work, we show that retrotransfer mediated by the IncP9 TOL pWW0 plasmid occurs in the rhizosphere, a niche in which the continuous supply of nutrients via root exudates allows cells to reach a high density. This suggests that this unusual sexual fertilization may be of great importance in lateral gene transfer. We also show that retrotransfer of DNA seems to require co-integration of the plasmid and the host chromosome and subsequent resolution, because a TOL plasmid with a mutation in the tnpR gene, encoding the resolvase of the Tn 4653 of the TOL plasmid, was self-transferred between Pseudomonas strains, but unable to mobilize chromosome.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.     

Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.     

Structural plasmid evolution as a result of coupled recombinations at bom and cer sites

We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.     

Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue

Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools. In many bacteria these extrachromosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult. Here we describe a system that will allow the rescue of any circular DNA (plasmid or phage) using an in vitro transposition system to deliver both a selectable marker (kanamycin) and an Escherichia coli plasmid origin of replication. In this study, we demonstrate the rescue of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium. To evaluate the host range of the rescued plasmids, we have examined their ability to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids. In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes. Using this approach, we identified a putative conjugative relaxase, suggesting this myco-bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.     

Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE

Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix α3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central ～3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.     

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ～9 kDa and ～30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.     

Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required.

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.     

Cloning and nucleotide sequence of a gene upstream of the eaeA gene of enterohemorrhagic Escherichia coli O157:H7

Abstract Conjugative, thermosensitive shuttle plasmids capable of transfer from Escherichia coli to Mycobacterium smegmatis were constructed. They contain both an E. coli replicon and a thermosensitive derivative of the pALS000 mycobacterial replicon. Using a temperature shift protocol, the conjugative plasmid, pJAZll was used to deliver the transposon Tn 611 from E. coli into the chromosome of M. smegmatis . Analysis of transconjugants revealed the random insertion of the transposon.     

Gene replacement and expression of foreign DNA in mycobacteria.

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.     

Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high. The recA function of both donor and recipient cells was required in the transfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid. This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.     

Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation

Hybrids of the Streptomyces coelicolor conjugative plasmid SCP2* and the Mycobacterium plasmid pAL5000 were transferred from Streptomyces coelicolor or Streptomyces lividans to Mycobacterium smegmatis mc2155 in plate crosses. Inactivation of the SCP2* transfer function did not prevent or reduce plasmid transfer. This transfer was DNase I sensitive and thus involved release of DNA from Streptomyces, followed by transformation of M. smegmatis. M. smegmatis growing on specific solid media was also transformed by pure CCC and linear plasmid DNA. Small plasmids were taken up intact but large plasmids suffered deletions. Competence developed within 24 h of incubation at 30 degrees C or 37 degrees C, and up to 400 transformants were obtained per microg of CCC plasmid DNA. Transformation frequencies were higher when M. smegmatis was co-cultivated with plasmid-free Streptomyces, but unaffected by resident homologous sequences or inactivation of recA in M. smegmatis. Spontaneous transformation was also observed with a circular Streptomyces transposable element which inserted into chromosomal sites. Transformative plasmid transfer was also shown to occur between M. smegmatis strains. This is the first report of non-artificially induced, spontaneous plasmid transformation in Mycobacterium.     

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.     

Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli

We used the LacO/GFP-LacI system to label and visualize the IncP beta plasmid R751 fluorescently during conjugative transfer between live donor and recipient bacteria. Comparisons of R751 in conjugative and non-conjugative conditions have allowed us to identify key localizations and movements associated with the initiation of conjugative transfer in the donor and the establishment of R751 in the recipient. A survey of successful mating pairs demonstrates that close physical contact between donor and recipient bacteria is required for DNA transfer and that regions of intimate contact can occur at any location on the donor or recipient cell membrane. The transferred DNA is positioned at the characteristic centre or quarter-cell position after conversion to a double-stranded molecule in the recipient cell. Initial duplication of plasmids often results in an asymmetric distribution of plasmid foci. Symmetric localization (either at centre or at 1/4 and 3/4 cell lengths) occurs only after a significant lag, presumably reflecting the time required to synthesize the plasmid-encoded partitioning proteins.