Annu. Rev. Microbiol. 1999. 53:217–44 Copyright c 1999 by Annual Reviews. All rights reserved.

DNA UPTAKE IN BACTERIA
David Dubnau
Public Health Research Institute, New York, NY 10016; e-mail: dubnau@phri.nyu.edu
Annu. Rev. Microbiol. 1999.53:217-244. Downloaded from www.annualreviews.org by Copenhagen University on 11/15/10. For personal use only.

Key Words competence, transformation, DNA transport, type-2 secretion, pilus formation s Abstract Natural competence is widespread among bacterial species. The mechanism of DNA uptake in both gram-positive and gram-negative bacteria is reviewed. The transformation pathways are discussed, with attention to the fate of donor DNA as it is processed by the competent cell. The proteins involved in mediating various steps in these pathways are described, and models for the transformation mechanisms are presented. Uptake of DNA across the inner membrane is probably similar in grampositive and gram-negative bacteria, and at least some of the required proteins are orthologs. The initial transformation steps differ, as expected, from the presence of an outer membrane only in the gram-negative organisms. The similarity of certain essential competence proteins to those required for the assembly of type-4 pili and for type-2 protein secretion is discussed. Finally several hypotheses for the biological role of transformation are presented and evaluated.

CONTENTS

Definitions and Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Uptake Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of DNA in Gram-Positive Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of DNA in Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competence Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Receptor Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competence Proteins Related to Those Involved in Type-4 Pilus Assembly and Secretion: The PSTC Proteins . . . . . . . . . . . . . . . . . . . . . . . . Role of the PSTC Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Competence Nuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competence Proteins Required for Transport . . . . . . . . . . . . . . . . . . . . . . . . . Additional Competence Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Energetics of DNA Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Models for DNA Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Gram-Positive Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Gram-Negative Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Use is Competence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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DUBNAU

DEFINITIONS AND SCOPE
Natural competence is a genetically programmed physiological state permitting the efficient uptake of macromolecular DNA. It is distinct from artificial transformation involving electroporation, protoplasts, and heat shock/CaCl2 treatment. This review deals only with natural competence. Recombination is not discussed. In many bacteria, competence is highly regulated, and much research has been devoted to exploring the complex control mechanisms involved. This regulatory work is not included here, and discussion is limited to the mechanism of DNA uptake and to the evolutionary significance of competence.

THE UPTAKE PATHWAY

In gram-positive bacteria, DNA must pass through the cell wall and the cytoplasmic membrane. In gram-negative bacteria, DNA must also traverse the outer membrane. Additional steps must therefore be involved in the gram-negative transformation systems, and the initial interaction of DNA with the cell surface must be different in the two types of bacteria. We begin with a description of the transformation pathway in the gram-positive organisms Bacillus subtilis and Streptococcus pneumoniae and then describe the gram-negative systems, with emphasis on Neisseria gonorrhoeae and Haemophilus influenzae. These initial descriptions are restricted to the fate of donor DNA. Discussion of the proteins involved is presented later. A summary of the transformation pathways is provided in Figure 1. Because these pathways were described in the earlier literature and have been extensively reviewed (32, 61, 76, 88, 128), they are presented briefly here.

Uptake of DNA in Gram-Positive Bacteria

Binding of DNA The first step in transformation is the binding of doublestranded DNA to the cell surface. In B. subtilis it has been estimated that there are ∼50 binding sites per competent cell (36, 126). Binding occurs with no detectable lag (36, 81) and without base sequence preference. Immediately after binding, intact double-stranded DNA can be recovered (38). In S. pneumoniae there also appears to be no base sequence specificity for transformation. It has been determined that there are 33–75 uptake sites per colony-forming unit in this organism (45). In both organisms the mass of DNA bound to the cell is proportional to the molecular size of the DNA (33, 69); binding occurs to a fixed number of sites with a probability that is independent of molecular weight. Fragmentation Within 30 sites of binding to B. subtilis, double-stranded DNA fragments are recovered that have been produced by limited cleavage of the initially bound donor molecules (7, 33, 35). These fragments are still completely accessible to added DNase. When either phage T7 DNA or higher-molecular-weight chromosomal donor DNA was used, a population of molecules with a number

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Annu. Rev. Microbiol. 1999.53:217-244. Downloaded from www.annualreviews.org by Copenhagen University on 11/15/10. For personal use only.

Transport In this review the term transport refers to the process of uptake across the (inner) cell membrane. subtilis that the distance between binding/fragmentation sites and the stiffness of the DNA determine the distance between cuts (32). 71. As the size of donor DNA increases. pneumoniae the nicking step may be concomitant with binding (70). pneumoniae. Rev. Cell-surface cleavage may therefore serve to generate DNA ends near cellular uptake sites. fragmentation on the cell surface has also been reported (70. Single ? Annu. single-strand nicks occur first. 1999. It has been suggested for B. 38). somewhat less than for B. average molecular size of ∼18 kilobases (kb) was recovered from the cell surface (33. and that between double-strand breaks is about the same (99).53:217-244. precursor single-stranded donor DNA can be recovered from transformed cells. For personal use only. 44. In both B. 98).5 kb (35). . Homologous single-stranded donor DNA integrates to form a heteroduplex consisting of base-paired donor and recipient strands (11. the search time needed to encounter these ends might place an unacceptable kinetic limitation on transformation. It follows that. It is not known whether nicking precedes double-strand cleavage in B. subtilis (28. In S. 72). it becomes less likely that binding will occur very close to a pre-existing end. Microbiol. In another study the estimate for the size of the surface-localized donor fragments was 13. 132). The average length between nicks is 6 kb. subtilis.org by Copenhagen University on 11/15/10. and double-strand breaks occur subsequently. In this organism. It is usually assumed that uptake proceeds from a newly formed end and not from pre-existing ends. if uptake were to proceed only from pre-existing ends. pneumoniae (68. 35. subtilis. 106) and S. In S.annualreviews.DNA UPTAKE IN BACTERIA 219 Figure 1 Transformation pathways in gram-positive (Bacillus subtilis and Streptococcus pneumoniae) and gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae) bacteria. Downloaded from www.

Downloaded from www. These products consist of 5 -nucleotides. subtilis. 106). This indicates that DNA is taken up in a linear fashion. the internalized single strands. bound donor chromosomal DNA becomes inaccessible to added DNase (81). In B. . However. acid-soluble degradation products appear in the medium (35). Microbiol. The nature of the lag preceding transport of a single genetic marker is unclear.5–10 kb (34. 43). Rev. after a lag of 1 to 1. and the non-covalently bound and final integrated donor segments is satisfying. the lag measured at this temperature is considerably longer (2.annualreviews. An unligated complex in which the donor and recipient moieties are stabilized only by base pairing has been detected (6.5 min) (130).220 DUBNAU strands are transported across the membrane. This compares reasonably with the length of the double-stranded fragments recoverable from the cell surface (13. 131). This correspondence is consistent with the hypothesis that these various molecular forms are related as precursors and products and that they are formed from one another in the order listed. and the integrated donor fragments (29. The average size of the final integrated donor DNA was 8.53:217-244. although it must include the time needed for both cleavage and for transport across the membrane of the minimal segment adequate for recombination. Based on this data and on newer sequence information. This contrasts with findings in other transformation systems in which transport occurs with 3 –5 polarity. we estimate that DNA passes into the cytosol at the rate of ∼180 nucleotides/s at 28◦ C. single-stranded DNA of donor origin can be recovered from lysed cells (28. The average length of these single-stranded fragments is 6–15 kb (28). and free bases. subtilis takes up single strands with either 5 –3 or 3 –5 polarity (139). The internalized strands interact efficiently with complementary sequences in the recipient chromosome to yield heteroduplex DNA. 1999. subtilis. ? Annu. the internalized single strands. These apparently concomitant events indicate that the single-stranded DNA has been transported into the cytosol. 37). It has been reported that B. Kinetic analysis has established that this is the case for the surface localized fragments.5 min at 37◦ C.3 kb. which increases with the physical distance between the markers (130. which was the temperature used for the marker pair uptake experiment. As noted above. marker pairs require additional time. Without such evidence one or more of these forms could be the products of side reactions. single genetic markers become DNase resistant after a 1. For personal use only.5-min lag during the transformation of B. whereas the non-transported strand is degraded.to 1. The average size of the donor moiety in this transient complex was estimated as 8.5–18 kb). Linear uptake fits nicely with the existence of an aqueous channel for the transport of DNA across the membrane.org by Copenhagen University on 11/15/10. subtilis the size correspondence between the surface-localized fragments. These single strands have not yet formed stable base-paired complexes with the recipient chromosome. At the same time. nucleosides. This should take less than a minute at 28◦ C. 36). In B. Simultaneously with the appearance of internalized single-stranded DNA and the acquisition of DNase resistance. it is likely that the nuclease responsible for this degradative step is localized outside the membrane or within an aqueous channel. However. Because nucleotides cannot ordinarily pass freely across membranes.

double-stranded molecules were recovered from the cells. gonorrhoeae uptake pathways are closely related. influenzae and N. 27. 39. influenzae genome revealed an extended 29-base-pair (bp) consensus uptake sequence. and a similar mechanism may exist to introduce ends for subsequent transport across the inner membrane. again implying linear uptake (49). not all gram-negative competence systems display such specificity. It is believed that transport initiates at a second break opposite the initial single-strand nick (70). influenzae and N. no additional cleavage events were detected. Rev. perhaps because of the rapidity with which DNase resistance is acquired. Downloaded from www. 1999. After the acquisition of DNase resistance. has not been characterized directly in the H. 103). influenzae the number of active sites for DNA uptake is only 4–8 per competent cell (30). Uptake sequences are present at frequencies far greater than those expected randomly.53:217-244. It is tempting to conclude from these results that the degradation and transport processes are mechanistically coupled. These consist of short oligonucleotides (74). although this is not certain. The polarity of DNA entry in this organism is 3 –5 (92). In an important study carried out with radiolabeled linear and circular donor molecules. 94). Binding A binding step. corresponding to the initial irreversible attachment of DNA to the cell surface in a form accessible to DNase or to shearing forces. However. DNase Resistance DNase resistance. when a larger plasmid DNA (42 kb) was used. The uptake rate was estimated as 90–100 nucleotides/s at 31◦ C. 127). Another major difference between the gram-positive and -negative systems is the presence of an outer membrane in the latter.annualreviews. has a different interpretation ? . Fragmentation Circular 11. which had been linearized at random positions. 50. and one strand equivalent is released into the medium as low-molecular-weight products (74. When linear plasmid DNA was used. more extensive doublestrand cleavage was observed. 77. However. Linked pairs require more time for entry than do single markers.org by Copenhagen University on 11/15/10. Microbiol. 98). For personal use only. In H. gonorrhoeae (9). used in the gram-positive systems as an index of transport across the cytoplasmic membrane. Uptake sequences are often found as inverted repeats between genes. Inspection of the H. Single strands appear in the cells (68. gonorrhoeae systems.DNA UPTAKE IN BACTERIA 221 Annu. Efficient DNA uptake by these gram-negative bacteria requires the presence of a specific uptake sequence (25. acting as transcriptional terminators (129). M´ jean & Claverys e (93) found that the degradation of the non-transported strand proceeded with 5 –3 polarity and at approximately the same rate as the entry of the transported strand. subtilis in most respects. Transport of DNA in S. An uptake rate of 500–1000 nucleotides/s has been measured (61). This pattern is reminiscent of that observed with the gram-positive systems. Uptake of DNA in Gram-Negative Bacteria The H. pneumoniae is similar to that in B.5-kb plasmid DNA was used to transform N. Acinetobacter calcoaceticus is able to take up DNA from any source (87.

In H. These proteins and their individual roles are now described. . This would imply the existence of a degradation-transport-recombination protein complex associated with the inner membrane. influenzae. Rev. Transport In H. and an initial binding step resulting in DNase-sensitive association of DNA with the cell surface was identified (107). a particle was observed on the surface of competent cells (61). Microbiol. Acinetobacter Transformation The transformation of the gram-negative A. and it is likely that one strand is degraded during transport across the inner membrane (61) just as in the gram-positive systems. Recently a low level of single-stranded donor DNA was detected during gonococcal transformation (16). in A. an attempt is made to integrate this information with our knowledge of the transformation pathways to construct summarizing models for gram-positive and gram-negative transformation. These “transformasomes” were considered to be structures in which double-stranded donor DNA is sequestered early in the transformation pathway. several proteins mediate DNA binding to the cell surface. and transformasomes deserve re-evaluation. subtilis. pneumoniae. For personal use only.222 DUBNAU in the gram-negative systems. they exhibit <10−7 of the wild-type transformation frequency. transformation results in single-strand integration (101). Instead.org by Copenhagen University on 11/15/10. However free cytoplasmic single-stranded DNA of donor origin has not been detected in this organism. ComEA is encoded by the first ? Annu.53:217-244. Many are conserved in the gram-positive and gram-negative systems. The acquisition of DNase resistance in these systems is not concomitant with the conversion to single-stranded DNA. DNA RECEPTOR PROTEIN In B. DNase resistance probably corresponds to entry into the periplasmic compartment or into a specialized structure. 1999. Moreover.annualreviews. calcoaceticus in some respects resembles that of the gram-positive systems. Mutants lacking these proteins are completely deficient in transformation (53). It is not certain that this material is a precursor of integrated DNA. This important work has not been pursued in recent years. COMPETENCE PROTEINS Several genes and proteins required for DNA uptake have been characterized. Transport proceeds in a 3 –5 direction (8). Instead it is believed that only a short length of single-stranded DNA is present at a given time as the DNA crosses the inner membrane and searches out a complementary sequence in the recipient. Downloaded from www. as it does in S. After these descriptions. calcoaceticus double-stranded molecules are converted to single strands on transport (103). The degradation of the nontransported strand may be concomitant with transport and integration. It was concluded that this organism takes up DNA from any source (103). influenzae.

Non-polar null mutations in comEA eliminate DNA binding. These properties of ComEA imply that it is a DNA receptor for transformation. However.annualreviews.DNA UPTAKE IN BACTERIA 223 Annu. Analysis of non-polar mutants lacking each of the seven ComG proteins demonstrated that they are individually needed for binding (19). Microbiol. all proteins needed for pilus formation must also be required for twitching motility. ComEA possesses a single membrane-spanning domain near its N terminus and is an integral membrane protein with its C terminus outward (60). Proteins possessing similarity to the C-terminal DNA-binding domain of ComEA are widespread in nature. A purified protein lacking this motif had no detectable DNA-binding activity in the gel shift assay.org by Copenhagen University on 11/15/10. with an apparent Kd of ∼5 × 10−7 M. calcoaceticus in view of the similarity of its transformation pathway to that of the gram-positive organisms. In both assays. Rev. A ComEA ortholog has been detected in S. there is no evidence that these proteins are involved in transformation. Membrane proteins from competent cultures were tested for DNA binding in southwestern assays. In their absence. and the resulting protein was purified (109). 53. and. Thus ComEA has more than one function in transformation. Because the formation of a pilus is a requirement for twitching motility (57). gonorrhoeae (109). for the type-2 secretion pathway. 104). These proteins resemble a widespread group required in gram-negative bacteria for the assembly of type-4 pili. These mutants are still able to bind DNA about half as well as the wild type. For personal use only. An in-frame deletion in comEA results in the expression of a shortened protein that is detectable in western blots (60). a ComEA ortholog may not be involved. pneumoniae and shown to be essential for transformation (15. subtilis (1. ComEA is intimately associated with the cell wall.over single-stranded DNA was noted. ? . 109). a marked preference for double. 60). including those in H. open reading frame of the comE operon (54. We call them PSTC proteins (for pilus/secretion/twitching motility/competence) (Table 1). and it was shown that intact ComEA was capable of binding DNA. It will be interesting to see whether such a protein is needed by A. and for twitching motility (58). Downloaded from www. and binding occurred with no apparent sequence specificity. unpublished data). The His6-ComEA protein bound to double-stranded DNA in gel shift assays. A His6-tag was substituted for the membrane-spanning domain of ComEA. but they fail to transport DNA. 96). transformation was undetectable. COMPETENCE PROTEINS RELATED TO THOSE INVOLVED IN TYPE-4 PILUS ASSEMBLY AND SECRETION: The PSTC Proteins Another group of proteins encoded by the comG operon and by comC was shown to be required for DNA binding in B. presumably via its C-terminal domain because it can be chemically cross-linked to wall material in vivo (YS Chung & D Dubnau. influenzae and N. because DNA uptake in these organisms requires a specific sequence.53:217-244. The C terminus of ComEA shows similarity to other nucleic acid-binding proteins and contains a possible helix-turn-helix motif (60. 1999.

calcoaceticus. f Pullulanase secretion representing class 2 secretion. 105) XcpV (8. D. PulE (111) PilB (104) 2 ComGB (1) CglB (107) CilD2 (16) ComYB (92) PilG (141) PilC (54) N. gonorrhoeae. D. D. . b Streptococci. subtilis. c N. 105) XcpU (8. PilQ (32) ComE (140) a B.org by Copenhagen University on 11/15/10. aeruginosa representing type 4 pilus assembly. e A.d A.c H. 5 None N. Downloaded from www. sa Strep. pullulanase secretion and pilus formation Competence proteins N. i. Microbiol.b 1 ComGA (1) CglA (107) CilD1 (16) ComYA (92) PilT (146) PilB (54) N. For personal use only. ? d H. g Pilus assembly in P. D.annualreviews. Rev. 105) XcpW (8. influenzae. PulF (111) PilC (104) 3 ComGC (1) ComGD (1) ComGE (1) ComGG (1) CgC (107) CglD (107) ComYC (92) PilE (128) PilA (54) ComP (110) PulG (119) PulH (119) Pull (119) PulJ (119) XcpT (8. 224 TABLE 1 PSTC proteins required for competence. c.e Pulf Pilg DUBNAU Class B. g. 105) PulO (114) PilD (104) PulD (58) PilQ (93) 4 ComC (98) CilC (16) ComYD (92) PilD (47) ComE (140) N.Annu.53:217-244. 1999.

ComGB of B. multiple class-3 proteins are needed. twitching motility (141. ComC (18. exemplified by ComGA in B. secretion. One of these. subtilis class-1 PSTC protein (ComGA) is located as a peripheral membrane protein on the cytosolic face of the membrane (18). has been shown to be an ATPase (116). gordonii (90).org by Copenhagen University on 11/15/10. except for ComGG. some molecules of ? Annu. 15. 104). . 143). 104) as well as in pilus assembly (102). in which a single class-3 protein was detected in a search of the genome (D Dubnau. TrwD. and type-2 protein secretion (108). 15. consisting of the B. 123. 20). unpublished data). The third class consists of small proteins with conserved sequences at their N-termini (2–4. They are integral membrane proteins. Rev. pneumoniae (15. 107. The B. 90. 90. ComGE. consisting of membrane proteins apparently with three predicted membrane-spanning segments. 107. consists of membraneassociated proteins with consensus nucleotide-binding sites. as well as in competence in gram-positive (1. and ComGG) and two proteins from the streptococcal systems possess such conserved sequences (15. Four of the B. Class-1 proteins are also required for conjugation-related systems. In nearly all of the systems that have been studied. which is accessible to proteinase K only in inside-out vesicles (18).DNA UPTAKE IN BACTERIA 225 which is thought to require pilus retraction (12). and CglC from S. The ubiquity of PSTC genes and their use for DNA and protein transport and for pilus assembly in diverse species suggest an ancient mechanism. influenzae. subtilis class-3 pre-proteins (ComGC. and pilus assembly. 18. anchored by their single predicted N-terminal membrane-spanning segments (13. subtilis (1). gonorrhoeae is a member of this class and is required for transformation (124). The reverse is not true. as is an Acinetobacter protein (107). In the absence of ComC. ComGD. ComGD. An exception may be H. subtilis ComG proteins (ComGC. Members of this group have been implicated in transformation of both gram-negative (52. In general the conservation of sequence among the members of this class is restricted to the hydrophobic N-terminal domain. The four B. subtilis ComGC protein. Representatives of this class are required for competence. 104). 138). PSTC proteins fall into five classes. However members of one subgroup required for competence. PilT of N. are similar over their entire lengths. the pre-proteins are organized with their C-termini outside the membrane. 104. and ComGG) are processed by the peptidase. ComYC from S. 1999. Microbiol. one of them is the precursor of the major structural protein of the pilus. Class 1. 20). required for conjugation of the plasmid R388. Class-2 proteins have been implicated in pilus assembly (102) and type-2 secretion (108). 143) and gram-positive bacteria (15. The major pilin protein of N. subtilis exemplifies a second class. ComGE. 104) and gram-negative systems (52. Downloaded from www.53:217-244. which predates the divergence of gram-positive and -negative bacteria. 124). 90. On processing. which are discussed in turn below. gonorrhoeae (143) and Pseudomonas aeruginosa (141) is needed for twitching motility but not for pilus formation. resembling the cleavage sites of type-4 prepilin proteins. Only for type-4 pilus assembly are the functions of any of these proteins understood. 115.annualreviews. For personal use only.

91). unpublished data). Rev. by the addition of crude preparations of pili-containing PilC (119). An additional protein. it is not absolutely required for pilus production (120) and does not resemble any of the PSTC proteins described above. or pili across the outer membrane. is in a homodimer that is stabilized by a disulfide bond (18). in at least some cases. A search of the completed B. on the other hand. 23. The single cysteine residue of ComGC is apparently involved in vivo in an intramolecular disulfide bond. protein. PilC. Secretins are required for competence (31. Processing of the class-3 B. This requires the peptidase (ComC) and is probably dependent on processing. 100. 136). 136) but not in gram-positive transformation systems. but no effect of the pilC null mutation on twitching motility or pilus formation was observed. 20). Protein pIV. Other secretins form similar structures (10. These cleave the class-3 proteins at a site within the N-terminal conserved sequence and. Interestingly. composed of ∼14 identical subunits. Downloaded from www. Additional cross-linking experiments have shown that portions of both the mature and unprocessed ComGC are probably present as homodimers. 56. is required for competence in N. competence can be partially restored to a pilC null mutant by the addition of purified PilC protein or. Microbiol. gonorrhoeae (119).annualreviews. 119). Although PilC is pilus-associated. subtilis genome failed to indicate the presence of a secretin (D Dubnau. pilus assembly (31. which exhibit sequence similarities in their C-terminal domains (121). even better. 1999. These results strongly suggest that PilC functions on the cell surface. For personal use only. DNA binding to the A.226 DUBNAU ComGC. subtilis competence proteins is dependent on the peptidase ComC (18. Secretins are also involved in filamentous phage maturation (122) and in the so-called type-3 secretion systems (62). 66). These proteins are located in the outer membrane. where they form large multisubunit complexes (17. 65. In A calcoaceticus. ComGG. unpublished data). The class-5 PSTC proteins are the secretins. calcoaceticus cell surface was also reduced. a PilC ortholog has also been identified as a competence protein (83). Consequently about one fourth of each mature class-3 protein is peripherally associated with the outer face of the membrane. undergo translocation and are no longer located as integral membrane proteins (18. The fourth class of proteins in this group consists of membrane-localized peptidase/transmethylases. It is not surprising that they have been implicated in gram-negative (31. A search of the ? Annu. Secretins are almost certainly involved in permitting passage of phage particles. . transfer a methyl group from S-adenosyl methionine to the newly formed primary amino group at the N-terminus (133). 125). 20). pIV forms a cylindrical complex. and type-2 secretion (24). A portion of ComGG. and possibly ComGE.53:217-244. the beststudied secretin. In both organisms this protein is required for DNA uptake to a DNase-resistant state (83. In its absence. DNA. We have recently found that these proteins can be efficiently cross-linked in vivo to cell wall material (YS Chung & D Dubnau. ComGD. and another fourth of the total is released from the protoplast on removal of the cell wall. with a central pore (82).org by Copenhagen University on 11/15/10. is required for the maturation of the filamentous phages.

subtilis genome failed to reveal the presence of a PilC ortholog (D Dubnau. EndA is localized in the membrane and possesses an uncleaved signal sequence that probably serves to anchor the protein in the membrane. Southwestern blotting experiments with competent cell membrane proteins. Although binding of DNA to intact cells requires each of the ComG proteins. . The cell wall in B. and ComGG.53:217-244. In A. the EndA protein is required for transport and for degradation of the nontransported strand but not for binding to the cell surface (73). These two modes of action are not mutually exclusive because the construction of such a channel may require cell wall modification. is complex and quite thick (5). It was suggested that EndA is fixed asymmetrically near a pore and that degradation of one strand may occur by successive endonucleolytic cleavages as DNA moves past the active site of the nuclease (118). Knockout of the streptococcal PSTC loci confers severe transformation deficiencies (15. 105). the PSTC class 3 protein so far identified is also required for binding to the cell surface (107). 118). DNA must therefore breach both physical and electrostatic barriers. but it is not needed to introduce the initial nick (70). failed to detect any evidence that these are DNA-binding proteins (R Provvedi & D Dubnau. A COMPETENCE NUCLEASE In S.DNA UPTAKE IN BACTERIA 227 B. In fact EndA was ? Annu. calcoaceticus. These experiments indicate a role for the PSTC proteins in providing access to the ComEA receptor. pneumoniae. the cell wall contains an abundance of teichoic acid polyanions. For personal use only. subtilis. but has no known ortholog. Right side-out membrane vesicles prepared from competent cells are able to bind double-stranded DNA in a ComEA-dependent reaction (109). Microbiol. 19).annualreviews. unpublished data). EndA is an endonuclease that acts on both RNA and DNA (117). Alternatively the pilin-like proteins may form a tunnel that traverses the wall and offers access to incoming DNA. In addition to peptidoglycan. with its bulk facing outward (75. Downloaded from www. ComGF is also required for binding in B. 1999. each of the PSTC proteins is required for the initial binding of DNA to the cell surface (13. as in most gram-positive organisms. This mechanism is consistent with the evidence described above suggesting that entry and degradation are coupled (93). ROLE OF THE PSTC PROTEINS In B. ComGE. unpublished data). It is likely that the PSTC proteins play analogous roles in these systems. The ComG proteins may modify the wall to increase porosity and locally shield or remove negative charges. binding in the vesicle system is unaffected by their absence.org by Copenhagen University on 11/15/10. and gel shift assays with purified ComGC. 90. ComGD. Rev. 111. It may also be needed for cleavage opposite the initial nick on the cell surface. subtilis. subtilis.

COMPETENCE PROTEINS REQUIRED FOR TRANSPORT It was noted above that ComEA plays a part in transport in addition to its role as the DNA receptor protein. implying that ComFA exists in a homo. This complex topology and its role in DNA transport suggest that ComEC may form all or part of an aqueous channel.53:217-244. ComFA possesses a consensus nucleotide-binding sequence. Transformation is decreased about 1000-fold in null mutants of comFA ? Annu.org by Copenhagen University on 11/15/10. influenzae (22). gonorrhoeae (41). A search of the complete B. subtilis comF operon is also required for transport but not for binding (85). ComFA resembles members of the DEAD family of helicases and has a strong similarity to PriA (84). Downloaded from www. The first ORF of the B. It is likely that this mutation permitted entry into the periplasm but not into the cytosolic compartment. Rev. In N. 104). Microbiol. . Another protein encoded by the comE operon. subtilis and N. ComEC is predicted to be a polytopic membrane protein with >6 membrane-spanning segments. in contrast to PSTC mutations. 60). other components of the transport apparatus. H. subtilis genome sequence failed to reveal an EndA ortholog (D Dubnau. unlike all other known competence proteins in the gram-positive systems.annualreviews. An excess of ComFA over other competence proteins may result in the formation of aberrant non-functional complexes. gonorrhoeae a mutation in the comEC ortholog did not interfere with DNA binding or with the acquisition of DNase resistance. an Escherichia coli helicase that translocates along single-stranded DNA in the 3 –5 direction. which prevented DNA binding (40). pneumoniae (15. Overexpression of the wild-type ComFA protein also decreases DNA transport markedly (85). EndA is constitutively expressed and represents the major endonuclease of S. in association with uncharacterized proteins (118). It is worth pointing out that. It is possible that each competence system has recruited its own non-dedicated nuclease.228 DUBNAU shown to be present in a large membrane-localized complex. For personal use only. and its solubility properties suggest that it is an integral membrane protein (85). ComFA is accessible to proteinase K only from the cytoplasmic face of the membrane. unpublished data). by using the energy of ATP hydrolysis (80). ComFA may be a membrane-localized DNA transporter protein that hydrolyzes ATP and translocates DNA. pneumoniae (74). No protein with nuclease activity required for transport has been identified in other competence systems. gonorrhoeae proteins. ComFA is located in the membrane fraction of competent cells. namely an absence of DNA transport and no effect on binding (86). Interestingly some of these mutations confer dominant negative phenotypes. In the cell ComFA may be associated with ComEC and ComEA. ComEC. 1999.or heteromultimer. and N. ComEC orthologs have been shown to play essential roles in transformation of S. Point mutations in this sequence confer the same phenotype as the loss-of-function mutation. is required for transport but is dispensable for binding (53. consistent with a common role for the B.

DprA is another protein required for transformation in H.annualreviews. unpublished data). A comFlike operon preceded by a competence regulatory signal [a cin box (15)] was also detected in S. In its absence. as well as in S. the other competence proteins may be able to position the end of an incoming strand near the aqueous channel. DprA may be needed to transport DNA across the inner membrane. Entry might then occur inefficiently via diffusion. Downloaded from www. ? . unpublished data). and proteins with similarity to ComFA were also detected in a search of the incomplete N. although the nuclease is most likely located outside the membrane. pneumoniae (15). For personal use only. knockouts of other B. unpublished data). Another gene. influenzae (63. Microbiol. 79) [also identified as orfF (136)] in H. For instance. Por is located in the periplasm and is required for competence-associated changes in the protein composition of the membrane. transport and filament formation might still occur with reduced efficiency.53:217-244. and it is uncertain whether a ComFA equivalent is an essential competence protein in the gram-negative systems. SSB (CilA) has been shown to be a competence protein in S. ADDITIONAL COMPETENCE PROTEINS Por. pneumoniae (15). 1999. No data exist concerning the possible roles of any of these proteins in transformation. which prevents transport without a major effect on binding. 64). influenzae. 84). consistent with the appearance of the acid soluble products in the medium (35). These results suggest that transport may be necessary for degradation. influenzae (137). It is possible that SSB plays a supporting role in uptake. pneumoniae (21). as the 3 donor DNA terminus enters the cytosol. but transformation was severely depressed. meningitidis databases (D Dubnau. also prevent the release of acid-soluble products from radiolabeled donor DNA (R Provvedi & D Dubnau. subtilis. Rev. (53. the dprA mutant took up DNA into a DNase-resistant form as readily as the wild type. In Haemophilus. subtilis competence genes decrease transformation at least 107-fold. a periplasmic protein disulfide oxidoreductase. gonorrhoeae and N.DNA UPTAKE IN BACTERIA 229 Annu. subtilis (P Tortosa & D Dubnau. perhaps directionally biased by interaction with single-strand binding protein (SSB) in the cytosol. has been described in B. comFC (84). In the absence of ComFA. It is likely that Por is needed for the correct folding of one or more competence proteins. In the absence of SSB. DNA binding does not occur. facilitating transport and formation of a DNARecA filament before integration. In contrast.org by Copenhagen University on 11/15/10. The comFA and comEC null mutations and the in-frame deletion of comEA. is required for the transformation of H. influenzae (42). A PriA ortholog was identified in H. pneumoniae (15) and B. A deletion mutant lacking the 33 C-terminal amino acid residues exhibited 10% of the wild-type transformability. Transport may serve to drag the incoming DNA past the nuclease active site. The product of this gene resembles com-101 (78. SSB may bind. An SSB ortholog (CilA) is required for transformation in S. As noted above.

. It was also concluded that both the pH gradient and the membrane potential are required for DNA uptake (51). initial stages of genetic transformation proceeded despite the drastically lowered value of the intracellular phosphorylation potential” (51).org by Copenhagen University on 11/15/10. The addition of arsenate to the transformation medium halved the ATP pool but had no effect on transformation. Inactivation of tpc. Extracts of a tpc mutant exhibited slightly decreased murein hydrolase activity. In H. comL encodes a protein that is associated with and perhaps covalently attached to peptidoglycan (46).. Grinius therefore proposed that DNA binds to the cell surface. resulting in transformation deficiency and a cell separation defect (47). transformation is inhibited by a number of uncoupling reagents. It is difficult to reconcile this phenotype with the one observed in B. Rev.230 DUBNAU Annu.53:217-244. a knockout mutant of com-101 bound DNA as well as the wild type. because measurement of intracellular ATP was carried out with the bulk culture. No ComL ortholog is encoded by B. 1999. a lipoprotein. Two competence genes in N. This protein may affect a step that follows transport. It was concluded that the “. Inactivation of ComFC in B. It was suggested that ComL and Tpc might be murein hydrolases. another N. subtilis it requires the competent ? . subtilis. is sufficient to support viability but not competence. In S. No obvious ortholog of Tpc exists in the database. and the extent of inhibition paralleled the effect of these reagents on membrane potential (51). responsible for providing access for DNA through the wall (46. subtilis culture become competent for transformation. Single-strand transformation does not use a totally different transport pathway because in B. subtilis (D Dubnau. unpublished data). but it released much less donor DNA in acid-soluble form (78). this model does not readily accommodate the observation that single-strand transformation occurs efficiently in many systems. another study with inhibitors (140) has concluded that pH alone is required for DNA transport in B. It appears that the N-terminal half of ComL. subtilis decreases transformation only 5. releasing the protons. Complete loss of comL is lethal. However. Microbiol. gonorrhoeae gene. For personal use only. does not affect DNA binding. but a pleiotropic mutation was characterized that decreased both competence and cell volume. whereas only ∼10% of the cells in a B. subtilis. suggesting a proton symport mechanism for DNA uptake. it was proposed that degradation of one strand provides the driving force for introduction of the intact strand (118). ENERGETICS OF DNA UPTAKE In B. this conclusion must be questioned.to 10-fold (84). and decreases transport only slightly (85). forming a nucleoprotein complex that binds protons and acquires a positive charge. although a similar protein (HI0177) is encoded by H. is also pleiotropic. subtilis. influenzae. 47). Inhibition occurred at or before the transport step because the acquisition of DNase resistance was inhibited. Downloaded from www. influenzae (42). However. pneumoniae. gonorrhoeae may be involved in murein metabolism. However. The complex then electrophoreses across the membrane.annualreviews.

comGE. It draws on evidence taken from both S.53:217-244. These substrate protein molecules are delivered to the periplasm. the available information is indirect and subject to various interpretations. In the figure we depict the class-3 PSTC proteins encoded by comGC. it was concluded that both components of the proton motive force could drive DNA uptake (14). Downloaded from www. possibly by contributing structurally to the transport apparatus or perhaps by playing an integral role in the delivery process. requires that the substrate macromolecules be in particular flexible states—unfolded for proteins and single-stranded for DNA. containing ComEC and ComFA. MODELS FOR DNA UPTAKE The information presented above can be summarized in the form of working models that ascribe particular roles to some of the competence proteins. 1999. Double-stranded DNA binds to the C-terminal domain of membraneanchored ComEA. Although transformation requires the input of metabolic energy. ComEA also plays a role in transport. influenzae. In H. is proton motive force required to drive DNA transport directly or to maintain the active state of an essential competence protein? Annu. probably through waterfilled channels. it is useful to compare transformation to the type-2 secretion systems in gram-negative bacteria (110). The Gram-Positive Model Figure 2 presents a working model for the transformation process in gram-positive bacteria. comGD. The newly formed DNA terminus is then delivered to the membrane-associated transport apparatus. Because all the relevant experiments so far involve the action of inhibitors on whole cells. Before presenting these models. an unfolded substrate protein is transported by the Sec system across the inner membrane.DNA UPTAKE IN BACTERIA 231 state (D Dubnau. PSTC proteins appear to be required to provide access through the wall and outer membrane and therefore to fulfill similar roles in all of these systems. In type-2 secretion. Rev.org by Copenhagen University on 11/15/10.annualreviews. A nuclease then cleaves the bound DNA near the point of contact with ComEA. double-stranded DNA must be transported across the outer membrane in the reverse direction. unpublished data). Transport across the inner membrane. pneumoniae and B. For personal use only. no clear picture exists as to the proximal source of this energy. A nuclease that degrades the non-transported strand (EndA in S. During transformation of gram-negative bacteria. Conversion to single-stranded DNA and transport across the inner membrane ensues by using proteins that are functionally analogous to the Sec proteins. subtilis. although most of the information concerning the roles of individual proteins relies on the latter. pneumoniae) is presumably also part of this apparatus. where they are folded and then transported across the outer membrane. and comGG as composing a structure ? . Microbiol. For instance. In the transformation and secretion systems.

1999. ComEA is similar to the C-terminal domain of Kid. One possible model for the delivery step is shown in which ComEA conformation is altered so that the bound DNA can contact the nuclease. It is possible that these proteins serve instead to increase the porosity of the wall or that the individual ComG proteins play different roles. Rev.org by Copenhagen University on 11/15/10. We postulate that ComEC forms an aqueous pore in the membrane for this transport step. First. releasing acid-soluble products into the medium. whereas in S. For personal use only. subtilis. The delivery process deserves comment. One possibility is that the DNAbinding domain of ComEA is close to the transport machinery. Kid also possesses a myosin-like N-terminal domain with a nucleotide-binding site that is lacking in ComEA.annualreviews.53:217-244. The protein nomenclature is from Bacillus subtilis. which reside on the inner face of the ? Annu. pneumoniae it is encoded by endA. ComGA and ComFA. References and further details are provided in the text. The nuclease has not been identified in B. Microbiol. Double-stranded DNA binds to the receptor protein ComEA. The single-strand product of nuclease action then contacts the transporter protein ComFA. a human kinesin-like DNA-binding protein (135). The ComG proteins are depicted. A doublestrand cleavage event is followed by delivery to a nuclease (N) that degrades one strand. some increasing porosity and some composing a wall-traversing structure. but it is likely that a similar mechanism operates in Streptococcus pneumoniae. and the energy of ATP hydrolysis is used to drive the DNA into the cell. Two suggestive features are consistent with this idea. that traverses the wall.232 DUBNAU Figure 2 Transformation in gram-positive bacteria. permitting access of DNA to the ComEA receptor. Downloaded from www. However. . Another is that some movement is required to establish this proximity. forming a structure that provides access of DNA to ComEA through the wall.

For personal use only. The Gram-Negative Model Figure 3 presents a model for transformation in H. A second suggestive phenomenon is that of twitching motility. In all of the well-studied transformation systems. ComFA is a component of this machinery. access to a DNA receptor analogous to ComEA may occur within the proposed PSTC protein complex. and these binding energies may facilitate uptake. As noted above. 1999. It is interesting that just upstream from the DNA-binding domain of ComEA is a flexible sequence (QQGGGG). gonorrhoeae. the mutant cells were able to bind but not to transport DNA (60). to inner membrane transport. In N. Because disassembly of this structure and delivery of the bound DNA to the uptake machinery would be time dependent. 3). subtilis ComEC protein mediate transport across the inner membrane. The previous discussion regarding the possible roles of SSB and of PTSC structure disassembly applies also to gram-negative transformation. It is believed that this form of bacterial motion requires the assembly and disassembly of pili. Downloaded from www. influenzae and N. Microbiol. A similar sequence is conserved in the S. the integration step may occur rapidly. PilC and the secretin protein PilQ are needed for outer membrane transport. By analogy with the B. gonorrhoeae (40). ComA [in N. As DNA enters the cytosol it probably associates with SSB and RecA. subtilis results described above.org by Copenhagen University on 11/15/10. and that this permits ComEA to bend and thereby deliver DNA to the transport machine. Rev. It is possible that the proposed wall-traversing structure disassembles by an analogous process.DNA UPTAKE IN BACTERIA 233 Annu. Perhaps bending of ComEA delivers DNA to the uptake apparatus. The proposed murein hydrolases Tpc and ComL may aid access through the wall. influenzae (22)] orthologs of the B. preventing the accumulation of single strands.53:217-244. a ComEC ortholog is required for DNA transport. the PSTC proteins allow DNA passage across the wall and periplasm. The binding site of ComFA is known to be required for transport (86). pneumoniae ortholog. membrane (18. Double-strand cleavage by an unknown nuclease also occurs (9). These are polytopic membrane proteins and may form uptake pores (Figures 2. contain nucleotide-binding sites. perhaps mobilizing the energy of ATP hydrolysis for this purpose. transformation in A. 48). When this was deleted. gonorrhoeae the phenotypes associated with loss-of-function mutations in several competence genes have been analyzed (40. Perhaps one of these proteins drives a conformational change in ComEA that delivers DNA to the transport pore. calcoaceticus may be different. the latter to form a pore. 85). subtilis and probably in S.annualreviews. if not mechanistically. gonorrhoeae (41)] or Rec2 [in H. resembling that of the gram-positive bacteria. associated with type-4 pili. Conversion to single-stranded DNA and degradation of one strand equivalent are probably closely coupled in time. Because large amounts of free single-stranded DNA are not detected. pneumoniae as well (21). subtilis. this type of mechanism may explain the lag observed before the acquisition of DNase resistance in B. ? . In B. and an ortholog of ComGA has been implicated in the disassembly process (141). Because these proteins are needed for binding in N.

and DNA for genetic diversity. These hypotheses can be characterized as DNA as food. and as is usual with evolutionary arguments they are not easily testable and may not be mutually exclusive. PilC. It was proposed that competence evolved to permit the uptake of DNA as a food supply (113. For personal use only. The protein nomenclature is taken from the Neisseria gonorrhoeae system. A review published five years ago listed >40 naturally transformable bacterial species (88). 114). The DNA receptor proteins and the nuclease that degrades one strand have not been identified. It is believed that the pilin protein (PilE) forms a structure that traverses the murein layer and that remodeling of the wall for transformation requires the proteins ComL and Tpc. References and further details are provided in the text. The transporter protein (T) has not been identified. as well as uptake systems for the nucleolytic products. Double-stranded DNA enters the periplasm through an outer membrane (OM) pore consisting of the secretin protein PilQ. 1999. Downloaded from www. a ComEC ortholog. ? Annu. a pilus-associated protein. is required for this step. possibly formed by ComA. Rev.234 DUBNAU Figure 3 Transformation in gram-negative bacteria. WHAT USE IS COMPETENCE? Competence is widespread. What is the selective force that has shaped and maintained these elaborate systems in so many species? Several hypotheses have been advanced. The intact single strand is then transported across the inner membrane (IM) through an aqueous pore. subtilis possesses a powerful nonspecific nuclease that is secreted into the medium. as if the expression of this capability must be finely tuned to the needs of each organism. For instance B.annualreviews. Transformation requires more than a dozen proteins and is often exquisitely regulated. This would seem to provide an efficient route for the consumption of environmental nucleic acids.53:217-244. Microbiol. but this reviewer believes that this is not a major factor among the selective pressures that maintain the competence mechanism. .org by Copenhagen University on 11/15/10. DNA for repair. but the mechanism is likely to be similar in Haemophilus influenzae.

A search of the Neisseria genome revealed two additional examples of the Haemophilus uptake sequence. subtilis is induced as part of the competence regulon (55. and oxidative damage to DNA may be more likely. The third popular hypothesis proposes that transformation is a mechanism for exploring the fitness landscape. 142). . but transformation with a cloned fragment had the same effect on survival as total chromosomal DNA. Similar data were reported for H. Microbiol.DNA UPTAKE IN BACTERIA 235 Why would the elaborate transformation machinery evolve to meet this need. not to the damage itself. influenzae transformation uptake sequences. Rev. Foreign DNA had no effect. sequencing of the sodC gene revealed the presence of two H.org by Copenhagen University on 11/15/10. subtilis. Many examples of horizontal gene transfer exist. The repair hypothesis has been criticized because DNA-damaging agents do not induce competence (112). 1999. This was interpreted as evidence for the horizontal acquisition of sodC from Haemophilus spp. which is met by a simpler and more generally useful pathway? It should be pointed out that. Downloaded from www. It cannot be ruled out that competence has served such a function under some conditions. because one strand equivalent is released into the medium. This suggestion is supported by the finding that the DNA repair machinery of B. influenzae. However. 142). both associated with Haemophilus-like genes. and perhaps at some point in evolutionary history. Somehow a recombination event increased the survival rate. in some organisms. which form the transcriptional terminator of this virulence determinant (67). Induction of a DNA repair system before the appearance of DNA damage could provide a selective advantage. gonorrhoeae systems exhibit uptake specificity. influenzae and N. Lysed cells provide DNA that is taken up and used for the repair of otherwise lethal lesions. For personal use only. coli than of H. as B. raising important doubts about the validity of the earlier experiments with B. one of the ? Annu. its metabolism becomes more aerobic. Tellingly. influenzae. when competence is induced. Such an induction mechanism may respond to conditions in which DNA damage is likely to occur. Two instructive examples have been reported recently. indicating that repair was not targeted to the site of integration as predicted by the repair hypothesis (97). a wasteful mechanism. This does not suggest a food-gathering mechanism. The H. subtilis approaches stationary phase. 95.53:217-244. the addition of DNA (59. The sequence of sodC resembled that of the Haemophilus ortholog more closely than the sod gene of Escherichia coli. For instance. All genetic diversity ultimately derives from mutation.annualreviews. A second proposal is that transformation serves a function in DNA repair (59. 95. whereas other Neisseria genes were more similar to those of E. and it is likely that transformation has played a role. subtilis. Such preventive induction might repair damaged DNA before serious harm is done by the secondary introduction of double-strand breaks or the synthesis of toxic proteins. In N. This was interpreted as favoring the DNA repair hypothesis. the competence machinery discards half of the potential foodstuff. transformation was found to increase survival in populations UV-irradiated before. 89). but this does not seem plausible as a general selective force. meningitidis. In B. but recombination can generate new allelic combinations. but not after. such induction is not a strong prediction of the repair hypothesis.

and it was demonstrated that polymorphisms in these loci are at linkage equilibrium.236 DUBNAU Annu. meningitidis. a cause of human gastritis (134). Mattick JS. Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon. Alm RA.org by Copenhagen University on 11/15/10. Because transformation is the only known means of genetic exchange in this organism. Microbiol. Visit the Annual Reviews home page at http://www. J. Identification of a gene. Rev. pilV. on the other hand.AnnualReviews.53:217-244. preventing these bottleneck effects from reducing diversity. it is tempting to conclude that this explains the selection for competence. Mol. 22:161–73 3. it is likely that competence is responsible for its panmictic population structure. 171:5386–404 2. It was pointed out (134) that recombination can contribute to genetic diversity in two ways. This may be telling us that competence can serve a variety of needs and that each species has learned to use the ability to transport DNA to meet its particular requirements. 1989. NIH Grants GM57720 and GM43756 supported the work from our group. ACKNOWLEDGMENTS I thank all the members of our lab for helpful comments on the manuscript and especially for countless stimulating discussions. Downloaded from www. whose . perhaps illustrated by the two examples given here. corresponding Haemophilus sequences contained a Neisseria uptake sequence. Microbiol. 1996.annualreviews. Watson AA. For personal use only. transformation has apparently served to introduce new genes from another species. Because transformation has actually enabled genetic exchange in natural populations. In the Neisseria example. In fact the signals that induce competence are species-specific. required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. providing a plausible mechanism for the transfer of the associated DNA from Haemophilus to N. and the debate concerning the evolutionary role of transformation will continue. Breitling R. Alm RA. In H pylori.org LITERATURE CITED 1. Hallinan JP. 1999. Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX in- ? crease the similarity of type 4 fimbriae to the GSP protein secretion systems and pilY1 encodes a gonococcal PilC homolog. Frequent recombination will rapidly disperse an advantageous mutation to many genetic backgrounds. Bacteriol. The sequences of three DNA fragments from clinical strains of this organism were determined. But this remains speculative. Albano M. 1995. Mattick JS. Dubnau D. Another example of evolution by transformation was discovered in Helicobacter pylori. recombination may have served to prevent a reduction in diversity caused by selective sweeps (whereas selection for a mutation that increases fitness causes the loss of competing genotypes with their associated diversity) and founder effects associated with the initial spread of a small number of organisms to a new niche.

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John J. R. For personal use only. Hurst Aerotaxis and Other Energy-Sensing Behavior in Bacteria. Ermias D. Holden The Induction of Apoptosis by Bacterial Pathogens. Van Etten. David Dubnau Integrating DNA: Transposases and Retroviral Integrases. Russel H. Carl E. Golden Constructing Polyketides: From Collie to Combinatorial Biosynthesis. Bennett Giant Viruses Infecting Algae. Ronald Bentley.53:217-244. L. J. Susan S. Chandler Transmissible Spongiform Encephalopathies in Humans. Johnson In Vivo Genetic Analysis of Bacterial Virulence. K-E. Three-Dimensional Structures. Bird Intercellular Signaling During Fruiting-Body Development of Myxococcus xanthus . annulata .Annual Review of Microbiology Volume 53. Haren.org by Copenhagen University on 11/15/10. B. Zhongming Ge. Gosink DNA Uptake in Bacteria. Shimkets Clostridial Toxins as Therapeutic Agents: Benefits of Nature's Most Toxic Proteins. Downloaded from www. Meints Mechanisms for Redox Control of Gene Expression. T. Anne Roulston. W. J. James T. Bauer. Gad Glaser Wolbachia Pipientis : Microbial Manipulator of Arthropod Reproduction. Ton-Hoang. G. D. Keith Gull 1 43 71 103 129 155 189 217 245 283 315 353 389 411 447 495 525 551 577 629 Annu. J. Staley. Taylor. Taylor Circadian Rhythms in Cyanobacteria: Adaptiveness and Mechanism.annualreviews. Carl Hirschie Johnson. W. and Biotechnological Applications of Lipases. Branton The Cytoskeleton of Trypanosomatid Parasites. Eric A. Lawrence J. Breeuwer. 1999 CONTENTS Transformation of Leukocytes by Theileria and T. Rev. B. M. Barry L. Belay Bacterial Biocatalysts: Molecular Biology. Jaeger. Su L. . David W. M. Stouthamer. Chiang. Yvette Weinrauch. Mark S. Volker Heussler Addiction Modules and Programmed Cell Death and Antideath in Bacterial Cultures. Hanna Engelberg-Kulka. Richard C. Marcellus. Igor B. Arturo Zychlinsky Poles Apart: Biodiversity and Biogeography of Sea Ice Bacteria. Zhulin. Microbiol. Dijkstra. Dirk Dobbelaere. D. John J. Terry H. Diane E. 1999. Mekalanos. Philip E. James L. Reetz Contributions of Genome Sequencing to Understanding the Biology of Helicobacter pylori . Sylvie Elsen. A. Johnson Viruses and Apoptosis.