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nature genetics • volume 34 • may 2003 3

Lateral transfer of DNA in bacterial popu-lations occurs by three naturally occurringprocesses: transformation, transductionand conjugation. Harnessing theseprocesses to generate genetic toolshas allowed us to mobilize, cloneand express DNA; generate spe-cific mutants; and complementmutant strains. Transformationand transduction have had animportant role in shaping the for-mative years of Mycobacteriummolecular genetics; electropora-tion and phage vectors are nowroutinely used for DNA cloningand transfer in many mycobacte-ria, including pathogenicMycobacterium tuberculosis, thecausative agent of tuberculosis1–5.Not much was known, however,about the process of conjugativeDNA transfer between mycobac-terial strains, with only a fewreports of conjugation-likeprocesses in Mycobacteriumspecies in the early 1970s6–8. Theprocess described involved asym-metric transfer of multiple chro-mosomal auxotrophic markersfrom a donor to a recipient strain,suggesting that transfer was byconjugation. In the subsequenttwo decades, however, research onconjugation was not pursued,despite the development ofgenetic tools for mycobacteria.

In 1998, a report by Keith Der-byshire and colleagues revisited this earlywork9. Using an array of molecular tech-niques that were previously unavailable,Parsons et al.9 confirmed and extendedearly findings of the presence of conjugativeDNA transfer in strains of Mycobacteriumsmegmatis. They showed that the processrequired prolonged cell–cell contact andthat the transfer of chromosomal markers(in this case, recombined antibiotic resis-tance genes) was unidirectional. The latterobservation allowed designation of certainstrains as donors and others as recipients.

The absence of plasmids in M. smegmatisled the group to believe that the transferfunctions were chromosomally located—either as an integrated phage, transposon or

conjugative plasmid that had lost the abilityto excise, or as unique elements in the chro-mosome that were responsible for anunusual transfer mechanism. On page 80 ofthis issue, Jun Wang and colleagues10 showthat the latter is true.

All in the chromosomePursuing the results described in their1998 paper9, Wang et al.10 have nowshown that conjugative DNA transferin M. smegmatis is mediated by multi-ple, chromosomally located cis-acting

elements. Using a chromosomal DNAlibrary of a donor strain cloned in a non-transferable shuttle vector, cis-actingregions of the chromosome were identi-

fied by their ability to confertransferability to the vector. Usingone such ‘Tra+’ plasmid (and itsderivates) as a laboratory model,the authors have made a numberof interesting observations aboutthe transfer process. Surprisingly,no homologs of known transfer-related genes were found on theplasmid. Unlike typical conjugaltransfer (characterized extensivelyin enteric bacteria), in which aunique cis-acting oriT is recog-nized by plasmid-encoded pro-teins that initiate and completeDNA transfer, conjugation of thetest plasmid in M. smegmatis wasmediated by a number of cis-act-ing chromosomal sequences,called bom (for basis of mobility),and required extended regions ofhomology with the recipientchromosome (see figure).

Perhaps the most interestingfeature of this unique transferprocess was the requirement of afunctional recA gene in the recipi-ent. This was because the Tra+

plasmid was ‘rescued’ in the recip-ient not by a relaxase-mediated re-ligation11 but by gap repair usinghost chromosomal sequences as atemplate. In an interesting parallel,

monomeric plasmids containing clonedchromosomal DNA are rescued in a similarmanner in the naturally competent Bacil-lus12 (although in this case, the plasmidDNA was transferred to the recipient bynatural transformation and did not requirespecific DNA sequences) (see figure).

Extending the results obtained with thebom-containing plasmid, one could specu-late that in a similar fashion, multiple bomsequences on the M. smegmatis chromo-some ensure that all regions have an equalchance of transfer. After transfer, the

Conjugal rites of mycobacteriaApoorva Bhatt & William R. Jacobs, Jr.

Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USAe-mail: jacobsw@hhmi.org

Gene products involved in the naturally occurring process of bacterial conjugation are usually encoded on plasmids. A new studyshows that in Mycobacterium smegmatis, a unique conjugative transfer is mediated by multiple cis-acting sequences present onthe chromosome and requires host recombination functions.

a b c d

a b c d

a b c d

degradation at bom region

transferredplasmid

recipientchromosome

rescuedplasmid

(1) strand invasion(2) gap repair

Bom mediated conjugation. According to the model proposed on page 82the plasmid is linearized at bom and transferred to the recipient, wherethe exposed ends are subject to exonucleolytic degradation. The 3′ endsthen invade homologous regions in the host chromosome, and the plas-mid is rescued by gap repair using the host chromosomal sequences as atemplate (Fig. 3a on page 82). As a result, the plasmid acquires interveningsequences from the recipient chromosome. This attribute can be used tocapture large chromosomal fragments: flanking sequences (patternedregion in a) can be cloned in a bom+ plasmid and then transferred to therecipient. Plasmid rescue by gap repair should yield a plasmid with theintervening chromosomal segment (b).

a

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4 nature genetics • volume 34 • may 2003

donor DNA recombines with homologoussequences in the host chromosome, lead-ing to the formation of a recombinant.

Going mobileThe precise role of the bom sequences isnot yet clear; they may serve as sites fornicking or double-stranded breaks thatinitiate the transfer of DNA, or, analogousto chi sites, they may merely be recruit-ment sites for the gap repair machinery.The study shows that although bomsequences are essential for conjugation,they are not sufficient to mediate transferbecause recipients of bom-containingplasmids do not further transfer the plas-mid to a third strain. Thus, there are pre-sumably other elements in thechromosome that are involved in the con-jugation process. This will no doubt beone of the most exciting prospects for fur-ther studies. Indeed, experiments definingthe difference between donor and recipi-ent chromosomes may result in the identi-fication of such ‘conjugation factors’.

It is not known what initiates the forma-tion of a mating pair because no pili (likethose involved in F-plasmid transfer inEscherichia coli) have been detected in M.smegmatis. In the simplest scenario, one ormore ‘transfer factors’ present or activeonly in M. smegmatis donor strains mayinitiate transfer by cleavage at a bom site onthe chromosome followed by transfer ofthe linearized DNA into the host cell. InStreptomyces lividans, a single integratedcopy of tra (transfer gene of a conjugativeplasmid) was sufficient to mediate transfer

of chromosomal DNA but promoted effi-cient transfer of plasmids only when theycontained a cis-acting element named clt13.The dispensability of clt for chromosomaltransfer implied that either chromosomaltransfer occurred by a different mechanismor, analogous to multiple bom sites in M.smegmatis, the S. lividans chromosome hadits own clt sequences.

Shuffling the deckThe ability of M. smegmatis to transferchromosomal markers by conjugation hasprofound evolutionary implications: notonly may mycobacteria that co-inhabitsimilar environmental niches exchangechromosomal markers and enrich theirgenetic repertoire, but genes or mutationsresponsible for drug resistance may also betransferred by conjugation. This raises thequestion: do other mycobacteria, includ-ing pathogenic M. tuberculosis, also pos-sess a similar transfer mechanism? So far,antibiotic resistance in M. tuberculosis hasbeen attributed to accumulation of spon-taneous mutations14. Also, non-tubercu-lous mycobacteria have been found toacquire antibiotic resistance genes fromother species, although this evidence, basedon sequence homology, could best bedescribed as circumstantial15. The ability ofM. smegmatis to transfer chromosomalmarkers by conjugation has opened up thepossibility that antibiotic resistance genes(and virulence factors) could be transferredbetween strains. It is indeed intriguing (andworrying) to envisage lateral transfer of adrug resistance–associated spontaneous

mutation between M. tuberculosis cells ininfected lung tissue.

As in other bacteria, conjugation couldalso be developed as an important genetictool. It offers an alternative gene deliverystrategy for introducing DNA from M.smegmatis to M. tuberculosis, M. bovis andother pathogenic species. The data alsoindicate the potential of this novel systemto allow ‘capture’ of segments of the chro-mosome by a bom+ plasmid containingthe flanking sequences (see figure). Simi-lar to the use of generalized transduction,this unusual conjugation may also be use-ful for moving markers between strains.The work of Wang et al.10 is the first steptowards this goal. �1. Snapper, S.B. et al. Proc. Natl. Acad. Sci. USA 85,

6987–6991 (1988).2. Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T. &

Jacobs, W.R.Jr. Mol. Microbiol. 4, 1911–1919 (1990).3. Lee, M.H., Pascopella, L., Jacobs, W.R.Jr. & Hatfull,

G.F. Proc. Natl. Acad. Sci. USA 88, 3111–3115 (1991).4. Bardarov, S. et al. Proc. Natl. Acad. Sci. USA 94,

10961–10966 (1997).5. Bardarov, S. et al. Microbiology 148, 3007–3017

(2002).6. Mizuguchi, Y. & Tokunaga, T. Igaku To

Seibutsugaku 81, 201–205 (1970).7. Tokunaga, T., Mizuguchi, Y. & Suga, K. J. Bacteriol.

113, 1104–1111 (1973).8. Mizuguchi, Y., Suga, K. & Tokunaga, T. Jpn. J.

Microbiol. 20, 435–443 (1976).9. Parsons, L.M., Jankowski, C.S. & Derbyshire, K.M.

Mol. Microbiol. 28, 571–582 (1998).10. Wang, J., Parsons, L.M. & Derbyshire, K.M. Nat.

Genet. 34, 80–84 (2003).11. Lanka, E. & Wilkins, B.M. Annu. Rev. Biochem. 64,

141–169 (1995).12. Canosi, U., Iglesias, A. & Trautner, T.A. Mol. Gen.

Genet. 181, 434–440 (1981).13. Pettis, G.S. & Cohen, S.N. Mol. Microbiol. 13,

955–964 (1994).14. Musser, J.M. Clin. Microbiol. Rev. 8, 496–514 (1995).15. Pang, Y., Brown, B.A., Steingrube, V.A., Wallace,

R.J.Jr. & Roberts, M.C. Antimicrob. AgentsChemother. 38, 1408–1412 (1994).

A nuclear address with influenceM. Frances Shannon

Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australiae-mail: frances.shannon@anu.edu.au

The spatial organization of the interphase nucleus is related to the differentiation state of the cell and has a role in establishingtissue-specific patterns of gene expression. Investigation of a tissue-specific nuclear protein has identified a new mechanism ofcell-specific gene regulation that links nuclear architecture, chromatin structure and gene transcription.

Distinctly staining regions of hete-rochromatin and euchromatin can beseen in interphase nuclei using electronmicrographs. The pattern of stainingcan distinguish different cell types,

which have distinct patterns of chro-matin organization. During differentia-tion, changes in gene expression arethought to be modulated by locatinggenes that need to be silenced in or near

regions of heterochromatin and genesthat need to be active away from hete-rochromatin1. An additional level oforganization in the nucleus has individ-ual chromosomes confined to discrete

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