S PECIAL S ECTION GGE ETTTTI N
I NGGAACCRROOS SS STTHHE EMME EMMBBRRAANNE E
27. F. U. Hartl, S. Lecker, E. Schiebel, J. P. Hendrick, W. 52. M. A. Harmey, G. Hallermayer, H. Korb, W. Neupert, 77. B. Meusser, C. Hirsch, E. Jarosch, T. Sommer, Nat.
Wickner, Cell 63, 269 (1990). Eur. J. Biochem. 81, 533 (1977). Cell Biol. 7, 766 (2005).
28. R. Lill et al., EMBO J. 8, 961 (1989). 53. K. P. Kunkele et al., Cell 93, 1009 (1998). 78. R. K. Plemper, S. Bohmler, J. Bordallo, T. Sommer, D. H.
29. L. Brundage, J. P. Hendrick, E. Schiebel, A. J. M. Driessen, 54. W. Neupert, M. Brunner, Nat. Rev. Mol. Cell Biol. 3, Wolf, Nature 388, 891 (1997).
W. Wickner, Cell 62, 649 (1990). 555 (2002). 79. B. Tsai, C. Rodighiero, M. Lencer, T. A. Rapoport, Cell
30. D. Görlich, T. A. Rapoport, Cell 75, 615 (1993). 55. M. Eilers, G. Schatz, Nature 322, 228 (1986). 104, 937 (2001).
31. S. Panzner, L. Dreier, E. Hartmann, S. Kostka, T. A. 56. P. Jarvis, C. Robinson, Curr. Biol. 14, R1064 (2004). 80. C. A. Jakob et al., EMBO Rep. 2, 423 (2001).
Rapoport, Cell 81, 561 (1995). 57. S. A. Paschen et al., Nature 426, 862 (2003). 81. M. Molinari, V. Calanca, C. Galli, P. Lucca, P. Paganetti,
32. K. Uchida, H. Mori, S. Mizushima, J. Biol. Chem. 270, 58. N. Wiedemann et al., Nature 424, 565 (2003). Science 299, 1397 (2003).
30862 (1995). 59. C. Sirrenberg, M. F. Bauer, B. Guiard, W. Neupert, M. 82. Y. Oda, N. Hosokawa, I. Wada, K. Nagata, Science
33. A. Economou, J. A. Pogliano, J. Beckwith, D. Oliver, W. Brunner, Nature 384, 582 (1996). 299, 1394 (2003).
Wickner, Cell 83, 1171 (1995). 60. J. M. Herrmann, W. Neupert, R. A. Stuart, EMBO J. 83. B. N. Lilley, H. L. Ploegh, Nature 429, 834 (2004).
34. T. Yahr, W. Wickner, EMBO J. 19, 4393 (2000). 16, 2217 (1997). 84. Y. Ye, Y. Shibata, C. Yun, D. Ron, T. A. Rapoport,
35. F. Duong, EMBO J. 22, 4375 (2003). 61. J. Serek et al., EMBO J. 23, 294 (2004). Nature 429, 841 (2004).
36. B. van den Berg et al., Nature 427, 36 (2004). 62. B. C. Berks, Mol. Microbiol. 22, 393 (1996). 85. M. Knop, A. Finger, T. Braun, K. Hellmuth, D. H. Wolf,
37. K. S. Cannon, E. Or, W. M. Clemons Jr., Y. Shibata, T. A. 63. A. Rodrigue, A. Chanal, K. Beck, M. Muller, L.-F. Wu, EMBO J. 15, 753 (1996).
Rapoport, J. Cell Biol. 169, 219 (2005). J. Biol. Chem. 274, 13223 (1999). 86. M. Pilon, R. Schekman, K. Romisch, EMBO J. 16, 4540
38. T. Powers, P. Walter, Science 269, 1422 (1995). 64. U. Gohlke et al., Proc. Natl. Acad. Sci. U.S.A. 102, (1997).
39. P. J. Rapiejko, R. Gilmore, Cell 89, 703 (1997). 10482 (2005). 87. R. J. Lee et al., EMBO J. 23, 2206 (2004).
40. V. Siegel, P. Walter, Nature 320, 81 (1986). 65. S. J. Gould, G.-A. Keller, N. Hosken, J. Wilkerson, S. 88. K. U. Kalies, S. Allan, T. Sergeyenko, H. Kroger, K.
41. V. Siegel, P. Walter, Cell 52, 39 (1988). Subramani, J. Cell Biol. 108, 1657 (1989). Romisch, EMBO J. 24, 2284 (2005).
42. Y. Kida, K. Mihara, M. Sakaguchi, EMBO J. 24, 3202 66. D. McCollum, E. Monosov, S. Subramani, J. Cell Biol. 89. N. N. Alder, S. M. Theg, Cell 112, 231 (2003).
(2005). 121, 761 (1993). 90. R. S. Ullers et al., J. Cell Biol. 161, 679 (2003).
43. W. Hansen, P. D. Garcia, P. Walter, Cell 45, 397 67. P. A. Walton, P. E. Hill, S. Subramani, Mol. Biol. Cell 91. K. E. S. Matlack, B. Misselwitz, K. Plath, R. A. Rapoport,
(1986). 6, 675 (1995). Cell 97, 553 (1999).
44. W. Hansen, P. Walter, J. Cell Biol. 106, 1075 (1988). 68. J. McNew, J. Goodman, J. Cell Biol. 127, 1245 (1994). 92. K. Nishiyama, T. Suzuki, H. Tokuda, Cell 85, 71
45. J. Toyn, A. R. Hibbs, P. Sanz, J. Crowe, D. I. Meyer, 69. V. Dammai, S. Subramani, Cell 105, 187 (2001). (1996).
EMBO J. 7, 4347 (1988). 70. P. E. Purdue, P. B. Lazarow, Annu. Rev. Cell Dev. Biol. 93. W. M. Clemons Jr., J.-F. Menetret, C. W. Akey, T. A.
46. B. C. Hann, P. Walter, Cell 67, 131 (1991). 17, 701 (2001). Rapoport, Curr. Opin. Struct. Biol. 14, 390 (2004).
Downloaded from http://science.sciencemag.org/ on January 2, 2021
47. S. C. Ogg, M. A. Poritz, P. Walter, Mol. Biol. Cell 3, 71. S. T. South, F. Baumgart, S. J. Gould, Proc. Natl. Acad. 94. N. Wiedemann, A. E. Frazier, N. Pfanner, J. Biol.
895 (1992). Sci. U.S.A. 98, 12027 (2001). Chem. 279, 14473 (2004).
48. N. D. Ulbrandt, J. A. Newitt, H. D. Bernstein, Cell 88, 72. V. I. Titorenko, R. A. Rachubinski, J. Cell Biol. 150, 95. J. Luirink, I. Sinning, Biochim. Biophys. Acta 1694, 17
187 (1997). 881 (2000). (2004).
49. G. J. Phillips, T. J. Silhavy, Nature 359, 744 (1992). 73. D. Hoepfner, D. Schildknegt, I. Braakman, P. Philippsen, 96. Work in the authors’ laboratories was supported by grants
50. D. Schuenemann et al., Proc. Natl. Acad. Sci. U.S.A. H. F. Tabak, Cell 122, 85 (2005). from the NIH and (for R.S.) by the Howard Hughes Medical
95, 10312 (1998). 74. T. Sommer, S. Jentsch, Nature 365, 176 (1993). Institute. We thank R. Lesch for preparing the figures and T.
51. M. A. Harmey, G. Hallermayer, W. Neupert, in Genetics 75. M. M. Hiller, A. Finger, M. Schweiger, D. H. Wolf, Pugsley, R. Gilmore, and T. Rapoport for critical suggestions.
and Biogenesis of Chloroplasts and Mitochondria, T. Science 273, 1725 (1996).
Bucher et al., Eds. (North Holland Publishing, Amster- 76. A. A. McCracken, J. Brodsky, J. Cell Biol. 132, 291
dam, 1976), pp. 813–818. (1996). 10.1126/science.1113752
REVIEW
The Ins and Outs of DNA Transfer in Bacteria
Inês Chen,1 Peter J. Christie,2* David Dubnau1*
Transformation and conjugation permit the passage of DNA through the bacterial genome plasticity over evolutionary history,
membranes and represent dominant modes for the transfer of genetic information and they are largely responsible for the rapid
between bacterial cells or between bacterial and eukaryotic cells. As such, they are spread of antibiotic resistance among patho-
responsible for the spread of fitness-enhancing traits, including antibiotic resistance. genic bacteria (3, 4).
Both processes usually involve the recognition of double-stranded DNA, followed by
the transfer of single strands. Elaborate molecular machines are responsible for Bacterial Transformation
negotiating the passage of macromolecular DNA through the layers of the cell Naturally transformable bacteria acquire a
surface. All or nearly all the machine components involved in transformation and physiological state known as ‘‘competence’’
conjugation have been identified, and here we present models for their roles in DNA through the regulated expression of genes for
transport. protein components of the uptake machinery.
Natural transformation has been most studied
In bacteria, transformation and conjugation transport phenomena in bacteria, such as the in Bacillus subtilis, Streptococcus pneumoniae,
usually mediate the transport of single- passage of DNA through the bacterial Neisseria gonorrhoeae, and Haemophilus
stranded DNA (ssDNA) across one or more division septa and those carried out by many influenzae. These and other competent bacteria
membranes. Transformation involves the bacteriophages (1), involve the movement of use similar proteins for DNA uptake, with
uptake of environmental DNA, whereas double-stranded DNA (dsDNA) and will not few differences between species. An in-
conjugation permits the direct transfer of be discussed here. Transformation and con- teresting exception is Helicobacter pylori,
DNA between cells (Fig. 1). Other DNA- jugation probably evolved for the acquisition which uses a conjugation-like system for
of fitness-enhancing genetic information, but transformation (5). Here, we will discuss the
1
Public Health Research Institute, 225 Warren Street, other mutually nonexclusive theories posit DNA uptake systems of B. subtilis and N.
Newark, NJ 07103, USA. 2Department of Micro-
biology and Molecular Genetics, University of Texas
that transformation might have evolved to gonorrhoeae as representative of those in
Medical School at Houston, Houston, TX 77030, USA. provide templates for DNA repair or to Gram-positive and -negative bacteria, re-
*To whom correspondence should be addressed.
supply nutrition for bacteria (2). Today, both spectively (Fig. 1A). The main distinction
Email: (P.J.C.); dubnau@ processes are recognized as important mech- between these cell types is that Gram-
phri.org (D.D.) anisms for horizontal gene transfer and negative bacteria are enclosed by cytoplas-
1456 2 DECEMBER 2005 VOL 310 SCIENCE www.sciencemag.org
, GETTING ACROSS THE MEMBRANE
S PECIAL S ECTION
and in some cases beyond the cell surface
A Transformation B Conjugation (19–22).
Gram Positive Gram Negative Gram Positive Gram Negative In B. subtilis, the ComG proteins neces-
Surface Mpf Conjugative sary for DNA binding (23) include the
adhesins channel pilus
AAAþ ATPase (ComGA), polytopic mem-
Binding Binding and
fragmentation G- brane protein (ComGB), major pre-pilin–like
G+
G+ Mating Fungus protein (ComGC), and three minor pre-pilin
G- junction Plant
Uptake into
Human proteins (ComGD, ComGE, and ComGG)
Fragmentation
periplasm (Fig. 2). The pre-pilin proteins integrate into
Substrate
processing
and transport
the cytoplasmic membrane, and when
processed by the peptidase ComC, these
Transport Transport
Plasmid
subunits translocate to the exterior of the
regeneration and
cell dissociation
membrane (24). Recently, a polymeric com-
plex dependent on the ComG proteins has
Fig. 1. Comparison of DNA processing and transfer during transformation and conjugation. (A) In been detected on the exterior of the membrane
transformation, dsDNA substrates are converted to single-stranded transfer intermediates for (25). This structure, termed a competence Y-
transport across the cytoplasmic membrane. (B) For conjugation, surface adhesins or conjugative pilus, consists of processed ComGC molecules
pili mediate donor-target cell contacts. Initial reactions involve the formation of a relaxase–T-DNA joined to one another by disulfide bonds and
transfer intermediate (green dot joined to black line) and tight mating junctions. Substrate transfer by additional noncovalent interactions. The
is probably mechanistically conserved in bacteria, although Gram-negative systems can deliver
substrates, including proteins (green dots), to phylogenetically diverse target cells (77–80). competence Y-pilus ranges in sizes cor-
responding to 40 to 100 subunits and, on the
basis of length estimates for a secretion Y-pilus
Downloaded from http://science.sciencemag.org/ on January 2, 2021
mic and outer membranes, with an inter- nuclease-resistant as it passes through the (22) and type IV pili (26), the competence Y-
vening periplasmic space and thin layer of outer membrane (Fig. 1A). This step requires pilus is long enough to traverse the periplasm
peptidoglycan (È3 to 7 nm) (6). Gram- the presence of a secretin protein (16). Se- and cell wall (È55 nm) (7).
positive bacteria lack an outer membrane, cretins form stable, donut-like multimers in N. gonorrhoeae produces type-4 pili, and
and their cytoplasmic membrane is sur- the outer membrane, with an aqueous central many proteins needed for pilus formation are
rounded by a È22-nm periplasmic space and cavity (17). Secretins are also components also required for DNA uptake and transfor-
a thick layer of peptidoglycan (È33 nm) (7). of type-4 pilus, filamentous phage-extrusion mation, leading to the assumption that pili
Initial interactions with the bacterial systems, and dedicated protein-secretion participate in DNA uptake. However, there is
surface. In both Gram-positive and Gram- systems, and they are also likely required evidence that two distinct structures exist in
negative bacteria, dsDNA interacts with the for conjugation. For transformation, DNA Neisseria, the type-4 pilus and a competence
competent cell surface by a process that is probably enters the periplasm through the Y-pilus, and that these structures apparently
not completely understood. DNA binds to secretin channel, although direct evidence compete for common components and mor-
competent B. subtilis cells in a state that is is lacking. The central cavity of the PilQ phogenetic proteins (27, 28).
resistant to centrifugal washing but susceptible dodecamer is 6.5 nm in diameter at its The growing secretion Y-pilus may act as
to added nucleases. ComEA, a membrane- widest point (17), adequate for the passage a piston, pushing substrate proteins through
bound dsDNA binding protein, is required of dsDNA (2.4 nm) or of a DNA-protein the secretin channel in the outer membrane
for transformation (8, 9). In the absence of complex. (18, 29, 30). Analogously, assembly and dis-
ComEA, 20% residual DNA binding still oc- The competence pseudopilus. Transforma- assembly of the competence Y-pilus may
curs in a competence-dependent manner (8). tion systems of Gram-negative and -positive contribute to DNA uptake by pulling DNA to
Similar results were observed in S. pneumoniae bacteria are made up of subunits with strik- the translocation machine in the cytoplasmic
(10), but the proteins responsible for this ing similarities to those needed for assembly membrane (Fig. 2). Repeated cycles of as-
residual binding remain unidentified in both of type-4 pili and type-2 secretion systems. sembly and disassembly would result in a low
species. In Gram-negative bacteria, dsDNA en- Type-4 pili are long and thin appendages concentration of maximal-length Y-pilus and
ters the periplasm, but in both Gram-negative that mediate a form of locomotion known as a broad size distribution, as observed for the
and -positive systems, a single strand of DNA twitching motility, which is powered by the B. subtilis competence Y-pili and the secre-
passes across the cytoplasmic membrane while extension and retraction of the pilus through tion Y-pili of Xanthomonas campestris (21).
its complement is degraded (Fig. 1A). DNA is assembly and disassembly. Type-2 secretion In single-molecule studies of DNA uptake in
taken up into the cytosolic space linearly (11), systems export folded-protein substrates B. subtilis (31), the rate of uptake (È80 base
and a free end is presumably required to initiate across the outer membrane through a secre- pairs sj1) was relatively constant with forces
the transport process. In B. subtilis, new termini tin channel. The conserved proteins for all up to 40 pN, without detectable pauses or
are provided by random cleavage events on the three systems include a cytoplasmic adenosine reversals. These features, unusual for molec-
cell surface, catalyzed by the integral mem- triphosphatase (ATPase) of the AAAþ ATPase ular motors that move along DNA, are
brane nuclease NucA (12). superfamily (ATPases associated with various similar to the force characteristics of type-4
Efficient DNA uptake in Neisseria and H. cellular activities), a polytopic membrane pilus retraction in N. gonorrhoeae (32). The
influenzae requires a species-specific DNA protein, a pre-pilin peptidase, and several proton motive force may be a source of
uptake sequence about 10 nucleotides long pilins or pilin-like proteins (18). In type-4 energy for DNA uptake; the rate of uptake
(13, 14). The genomes of these bacteria are pilus systems, these proteins mediate the decreases sharply with the addition of
enriched for their respective uptake se- assembly of the major pilin into the pilus uncoupling agents before any detectable
quences, favoring the uptake of homospe- fibers. Genetic manipulation, e.g., pilin over- decline in the ATP pool (31). Thus, the pro-
cific DNA (15). However, sequence-specific production, of a number of type-2 secretion ton motive force might directly drive the
binding receptors have not yet been identified. systems also results in the production of movement of the Y-pilus subunits into the
Secretins and uptake into the periplasm. pilus-like structures, termed pseudopili membrane, causing Y-pilus disassembly and
In Gram-negative bacteria, dsDNA becomes (Y-pili), that extend through the periplasm retraction.
www.sciencemag.org SCIENCE VOL 310 2 DECEMBER 2005 1457
I NGGAACCRROOS SS STTHHE EMME EMMBBRRAANNE E
27. F. U. Hartl, S. Lecker, E. Schiebel, J. P. Hendrick, W. 52. M. A. Harmey, G. Hallermayer, H. Korb, W. Neupert, 77. B. Meusser, C. Hirsch, E. Jarosch, T. Sommer, Nat.
Wickner, Cell 63, 269 (1990). Eur. J. Biochem. 81, 533 (1977). Cell Biol. 7, 766 (2005).
28. R. Lill et al., EMBO J. 8, 961 (1989). 53. K. P. Kunkele et al., Cell 93, 1009 (1998). 78. R. K. Plemper, S. Bohmler, J. Bordallo, T. Sommer, D. H.
29. L. Brundage, J. P. Hendrick, E. Schiebel, A. J. M. Driessen, 54. W. Neupert, M. Brunner, Nat. Rev. Mol. Cell Biol. 3, Wolf, Nature 388, 891 (1997).
W. Wickner, Cell 62, 649 (1990). 555 (2002). 79. B. Tsai, C. Rodighiero, M. Lencer, T. A. Rapoport, Cell
30. D. Görlich, T. A. Rapoport, Cell 75, 615 (1993). 55. M. Eilers, G. Schatz, Nature 322, 228 (1986). 104, 937 (2001).
31. S. Panzner, L. Dreier, E. Hartmann, S. Kostka, T. A. 56. P. Jarvis, C. Robinson, Curr. Biol. 14, R1064 (2004). 80. C. A. Jakob et al., EMBO Rep. 2, 423 (2001).
Rapoport, Cell 81, 561 (1995). 57. S. A. Paschen et al., Nature 426, 862 (2003). 81. M. Molinari, V. Calanca, C. Galli, P. Lucca, P. Paganetti,
32. K. Uchida, H. Mori, S. Mizushima, J. Biol. Chem. 270, 58. N. Wiedemann et al., Nature 424, 565 (2003). Science 299, 1397 (2003).
30862 (1995). 59. C. Sirrenberg, M. F. Bauer, B. Guiard, W. Neupert, M. 82. Y. Oda, N. Hosokawa, I. Wada, K. Nagata, Science
33. A. Economou, J. A. Pogliano, J. Beckwith, D. Oliver, W. Brunner, Nature 384, 582 (1996). 299, 1394 (2003).
Wickner, Cell 83, 1171 (1995). 60. J. M. Herrmann, W. Neupert, R. A. Stuart, EMBO J. 83. B. N. Lilley, H. L. Ploegh, Nature 429, 834 (2004).
34. T. Yahr, W. Wickner, EMBO J. 19, 4393 (2000). 16, 2217 (1997). 84. Y. Ye, Y. Shibata, C. Yun, D. Ron, T. A. Rapoport,
35. F. Duong, EMBO J. 22, 4375 (2003). 61. J. Serek et al., EMBO J. 23, 294 (2004). Nature 429, 841 (2004).
36. B. van den Berg et al., Nature 427, 36 (2004). 62. B. C. Berks, Mol. Microbiol. 22, 393 (1996). 85. M. Knop, A. Finger, T. Braun, K. Hellmuth, D. H. Wolf,
37. K. S. Cannon, E. Or, W. M. Clemons Jr., Y. Shibata, T. A. 63. A. Rodrigue, A. Chanal, K. Beck, M. Muller, L.-F. Wu, EMBO J. 15, 753 (1996).
Rapoport, J. Cell Biol. 169, 219 (2005). J. Biol. Chem. 274, 13223 (1999). 86. M. Pilon, R. Schekman, K. Romisch, EMBO J. 16, 4540
38. T. Powers, P. Walter, Science 269, 1422 (1995). 64. U. Gohlke et al., Proc. Natl. Acad. Sci. U.S.A. 102, (1997).
39. P. J. Rapiejko, R. Gilmore, Cell 89, 703 (1997). 10482 (2005). 87. R. J. Lee et al., EMBO J. 23, 2206 (2004).
40. V. Siegel, P. Walter, Nature 320, 81 (1986). 65. S. J. Gould, G.-A. Keller, N. Hosken, J. Wilkerson, S. 88. K. U. Kalies, S. Allan, T. Sergeyenko, H. Kroger, K.
41. V. Siegel, P. Walter, Cell 52, 39 (1988). Subramani, J. Cell Biol. 108, 1657 (1989). Romisch, EMBO J. 24, 2284 (2005).
42. Y. Kida, K. Mihara, M. Sakaguchi, EMBO J. 24, 3202 66. D. McCollum, E. Monosov, S. Subramani, J. Cell Biol. 89. N. N. Alder, S. M. Theg, Cell 112, 231 (2003).
(2005). 121, 761 (1993). 90. R. S. Ullers et al., J. Cell Biol. 161, 679 (2003).
43. W. Hansen, P. D. Garcia, P. Walter, Cell 45, 397 67. P. A. Walton, P. E. Hill, S. Subramani, Mol. Biol. Cell 91. K. E. S. Matlack, B. Misselwitz, K. Plath, R. A. Rapoport,
(1986). 6, 675 (1995). Cell 97, 553 (1999).
44. W. Hansen, P. Walter, J. Cell Biol. 106, 1075 (1988). 68. J. McNew, J. Goodman, J. Cell Biol. 127, 1245 (1994). 92. K. Nishiyama, T. Suzuki, H. Tokuda, Cell 85, 71
45. J. Toyn, A. R. Hibbs, P. Sanz, J. Crowe, D. I. Meyer, 69. V. Dammai, S. Subramani, Cell 105, 187 (2001). (1996).
EMBO J. 7, 4347 (1988). 70. P. E. Purdue, P. B. Lazarow, Annu. Rev. Cell Dev. Biol. 93. W. M. Clemons Jr., J.-F. Menetret, C. W. Akey, T. A.
46. B. C. Hann, P. Walter, Cell 67, 131 (1991). 17, 701 (2001). Rapoport, Curr. Opin. Struct. Biol. 14, 390 (2004).
Downloaded from http://science.sciencemag.org/ on January 2, 2021
47. S. C. Ogg, M. A. Poritz, P. Walter, Mol. Biol. Cell 3, 71. S. T. South, F. Baumgart, S. J. Gould, Proc. Natl. Acad. 94. N. Wiedemann, A. E. Frazier, N. Pfanner, J. Biol.
895 (1992). Sci. U.S.A. 98, 12027 (2001). Chem. 279, 14473 (2004).
48. N. D. Ulbrandt, J. A. Newitt, H. D. Bernstein, Cell 88, 72. V. I. Titorenko, R. A. Rachubinski, J. Cell Biol. 150, 95. J. Luirink, I. Sinning, Biochim. Biophys. Acta 1694, 17
187 (1997). 881 (2000). (2004).
49. G. J. Phillips, T. J. Silhavy, Nature 359, 744 (1992). 73. D. Hoepfner, D. Schildknegt, I. Braakman, P. Philippsen, 96. Work in the authors’ laboratories was supported by grants
50. D. Schuenemann et al., Proc. Natl. Acad. Sci. U.S.A. H. F. Tabak, Cell 122, 85 (2005). from the NIH and (for R.S.) by the Howard Hughes Medical
95, 10312 (1998). 74. T. Sommer, S. Jentsch, Nature 365, 176 (1993). Institute. We thank R. Lesch for preparing the figures and T.
51. M. A. Harmey, G. Hallermayer, W. Neupert, in Genetics 75. M. M. Hiller, A. Finger, M. Schweiger, D. H. Wolf, Pugsley, R. Gilmore, and T. Rapoport for critical suggestions.
and Biogenesis of Chloroplasts and Mitochondria, T. Science 273, 1725 (1996).
Bucher et al., Eds. (North Holland Publishing, Amster- 76. A. A. McCracken, J. Brodsky, J. Cell Biol. 132, 291
dam, 1976), pp. 813–818. (1996). 10.1126/science.1113752
REVIEW
The Ins and Outs of DNA Transfer in Bacteria
Inês Chen,1 Peter J. Christie,2* David Dubnau1*
Transformation and conjugation permit the passage of DNA through the bacterial genome plasticity over evolutionary history,
membranes and represent dominant modes for the transfer of genetic information and they are largely responsible for the rapid
between bacterial cells or between bacterial and eukaryotic cells. As such, they are spread of antibiotic resistance among patho-
responsible for the spread of fitness-enhancing traits, including antibiotic resistance. genic bacteria (3, 4).
Both processes usually involve the recognition of double-stranded DNA, followed by
the transfer of single strands. Elaborate molecular machines are responsible for Bacterial Transformation
negotiating the passage of macromolecular DNA through the layers of the cell Naturally transformable bacteria acquire a
surface. All or nearly all the machine components involved in transformation and physiological state known as ‘‘competence’’
conjugation have been identified, and here we present models for their roles in DNA through the regulated expression of genes for
transport. protein components of the uptake machinery.
Natural transformation has been most studied
In bacteria, transformation and conjugation transport phenomena in bacteria, such as the in Bacillus subtilis, Streptococcus pneumoniae,
usually mediate the transport of single- passage of DNA through the bacterial Neisseria gonorrhoeae, and Haemophilus
stranded DNA (ssDNA) across one or more division septa and those carried out by many influenzae. These and other competent bacteria
membranes. Transformation involves the bacteriophages (1), involve the movement of use similar proteins for DNA uptake, with
uptake of environmental DNA, whereas double-stranded DNA (dsDNA) and will not few differences between species. An in-
conjugation permits the direct transfer of be discussed here. Transformation and con- teresting exception is Helicobacter pylori,
DNA between cells (Fig. 1). Other DNA- jugation probably evolved for the acquisition which uses a conjugation-like system for
of fitness-enhancing genetic information, but transformation (5). Here, we will discuss the
1
Public Health Research Institute, 225 Warren Street, other mutually nonexclusive theories posit DNA uptake systems of B. subtilis and N.
Newark, NJ 07103, USA. 2Department of Micro-
biology and Molecular Genetics, University of Texas
that transformation might have evolved to gonorrhoeae as representative of those in
Medical School at Houston, Houston, TX 77030, USA. provide templates for DNA repair or to Gram-positive and -negative bacteria, re-
*To whom correspondence should be addressed.
supply nutrition for bacteria (2). Today, both spectively (Fig. 1A). The main distinction
Email: (P.J.C.); dubnau@ processes are recognized as important mech- between these cell types is that Gram-
phri.org (D.D.) anisms for horizontal gene transfer and negative bacteria are enclosed by cytoplas-
1456 2 DECEMBER 2005 VOL 310 SCIENCE www.sciencemag.org
, GETTING ACROSS THE MEMBRANE
S PECIAL S ECTION
and in some cases beyond the cell surface
A Transformation B Conjugation (19–22).
Gram Positive Gram Negative Gram Positive Gram Negative In B. subtilis, the ComG proteins neces-
Surface Mpf Conjugative sary for DNA binding (23) include the
adhesins channel pilus
AAAþ ATPase (ComGA), polytopic mem-
Binding Binding and
fragmentation G- brane protein (ComGB), major pre-pilin–like
G+
G+ Mating Fungus protein (ComGC), and three minor pre-pilin
G- junction Plant
Uptake into
Human proteins (ComGD, ComGE, and ComGG)
Fragmentation
periplasm (Fig. 2). The pre-pilin proteins integrate into
Substrate
processing
and transport
the cytoplasmic membrane, and when
processed by the peptidase ComC, these
Transport Transport
Plasmid
subunits translocate to the exterior of the
regeneration and
cell dissociation
membrane (24). Recently, a polymeric com-
plex dependent on the ComG proteins has
Fig. 1. Comparison of DNA processing and transfer during transformation and conjugation. (A) In been detected on the exterior of the membrane
transformation, dsDNA substrates are converted to single-stranded transfer intermediates for (25). This structure, termed a competence Y-
transport across the cytoplasmic membrane. (B) For conjugation, surface adhesins or conjugative pilus, consists of processed ComGC molecules
pili mediate donor-target cell contacts. Initial reactions involve the formation of a relaxase–T-DNA joined to one another by disulfide bonds and
transfer intermediate (green dot joined to black line) and tight mating junctions. Substrate transfer by additional noncovalent interactions. The
is probably mechanistically conserved in bacteria, although Gram-negative systems can deliver
substrates, including proteins (green dots), to phylogenetically diverse target cells (77–80). competence Y-pilus ranges in sizes cor-
responding to 40 to 100 subunits and, on the
basis of length estimates for a secretion Y-pilus
Downloaded from http://science.sciencemag.org/ on January 2, 2021
mic and outer membranes, with an inter- nuclease-resistant as it passes through the (22) and type IV pili (26), the competence Y-
vening periplasmic space and thin layer of outer membrane (Fig. 1A). This step requires pilus is long enough to traverse the periplasm
peptidoglycan (È3 to 7 nm) (6). Gram- the presence of a secretin protein (16). Se- and cell wall (È55 nm) (7).
positive bacteria lack an outer membrane, cretins form stable, donut-like multimers in N. gonorrhoeae produces type-4 pili, and
and their cytoplasmic membrane is sur- the outer membrane, with an aqueous central many proteins needed for pilus formation are
rounded by a È22-nm periplasmic space and cavity (17). Secretins are also components also required for DNA uptake and transfor-
a thick layer of peptidoglycan (È33 nm) (7). of type-4 pilus, filamentous phage-extrusion mation, leading to the assumption that pili
Initial interactions with the bacterial systems, and dedicated protein-secretion participate in DNA uptake. However, there is
surface. In both Gram-positive and Gram- systems, and they are also likely required evidence that two distinct structures exist in
negative bacteria, dsDNA interacts with the for conjugation. For transformation, DNA Neisseria, the type-4 pilus and a competence
competent cell surface by a process that is probably enters the periplasm through the Y-pilus, and that these structures apparently
not completely understood. DNA binds to secretin channel, although direct evidence compete for common components and mor-
competent B. subtilis cells in a state that is is lacking. The central cavity of the PilQ phogenetic proteins (27, 28).
resistant to centrifugal washing but susceptible dodecamer is 6.5 nm in diameter at its The growing secretion Y-pilus may act as
to added nucleases. ComEA, a membrane- widest point (17), adequate for the passage a piston, pushing substrate proteins through
bound dsDNA binding protein, is required of dsDNA (2.4 nm) or of a DNA-protein the secretin channel in the outer membrane
for transformation (8, 9). In the absence of complex. (18, 29, 30). Analogously, assembly and dis-
ComEA, 20% residual DNA binding still oc- The competence pseudopilus. Transforma- assembly of the competence Y-pilus may
curs in a competence-dependent manner (8). tion systems of Gram-negative and -positive contribute to DNA uptake by pulling DNA to
Similar results were observed in S. pneumoniae bacteria are made up of subunits with strik- the translocation machine in the cytoplasmic
(10), but the proteins responsible for this ing similarities to those needed for assembly membrane (Fig. 2). Repeated cycles of as-
residual binding remain unidentified in both of type-4 pili and type-2 secretion systems. sembly and disassembly would result in a low
species. In Gram-negative bacteria, dsDNA en- Type-4 pili are long and thin appendages concentration of maximal-length Y-pilus and
ters the periplasm, but in both Gram-negative that mediate a form of locomotion known as a broad size distribution, as observed for the
and -positive systems, a single strand of DNA twitching motility, which is powered by the B. subtilis competence Y-pili and the secre-
passes across the cytoplasmic membrane while extension and retraction of the pilus through tion Y-pili of Xanthomonas campestris (21).
its complement is degraded (Fig. 1A). DNA is assembly and disassembly. Type-2 secretion In single-molecule studies of DNA uptake in
taken up into the cytosolic space linearly (11), systems export folded-protein substrates B. subtilis (31), the rate of uptake (È80 base
and a free end is presumably required to initiate across the outer membrane through a secre- pairs sj1) was relatively constant with forces
the transport process. In B. subtilis, new termini tin channel. The conserved proteins for all up to 40 pN, without detectable pauses or
are provided by random cleavage events on the three systems include a cytoplasmic adenosine reversals. These features, unusual for molec-
cell surface, catalyzed by the integral mem- triphosphatase (ATPase) of the AAAþ ATPase ular motors that move along DNA, are
brane nuclease NucA (12). superfamily (ATPases associated with various similar to the force characteristics of type-4
Efficient DNA uptake in Neisseria and H. cellular activities), a polytopic membrane pilus retraction in N. gonorrhoeae (32). The
influenzae requires a species-specific DNA protein, a pre-pilin peptidase, and several proton motive force may be a source of
uptake sequence about 10 nucleotides long pilins or pilin-like proteins (18). In type-4 energy for DNA uptake; the rate of uptake
(13, 14). The genomes of these bacteria are pilus systems, these proteins mediate the decreases sharply with the addition of
enriched for their respective uptake se- assembly of the major pilin into the pilus uncoupling agents before any detectable
quences, favoring the uptake of homospe- fibers. Genetic manipulation, e.g., pilin over- decline in the ATP pool (31). Thus, the pro-
cific DNA (15). However, sequence-specific production, of a number of type-2 secretion ton motive force might directly drive the
binding receptors have not yet been identified. systems also results in the production of movement of the Y-pilus subunits into the
Secretins and uptake into the periplasm. pilus-like structures, termed pseudopili membrane, causing Y-pilus disassembly and
In Gram-negative bacteria, dsDNA becomes (Y-pili), that extend through the periplasm retraction.
www.sciencemag.org SCIENCE VOL 310 2 DECEMBER 2005 1457
