The question of why natural transformation exists remains the subject of ongoing debate. Three hypotheses are frequently discussed: ‘DNA for food’, ‘DNA for repair’, and ‘DNA for evolution’. These hypotheses are not necessarily mutually exclusive, but they are not equally supported by the data. This topic has recently been extensively reviewed elsewhere (Michod et al., 2008; Vos, 2009); however, due to the strong link between the induction of natural competence by environmental signals and the role transformation may play in bacteria, we summarize some of the major arguments below.
DNA for food
The role of DNA as a source of nutrients was initially proposed by Redfield (2001). She wrote, ‘the simplest model thus might be that the nutrients that transformation reliably provides pay the evolutionary bills (and have been responsible for the evolution of its regulation), and as a bonus the cell gets the occasional benefits of recombination and repair' (Redfield, 1993b). Among others, the main arguments put forward by Redfield for a role of transformation as ‘Genes for Breakfast’ were as follows: (1) DNA as food provides a direct short-term advantage, whereas a role of novel genes in evolution might be selected only in the long run. (2) H. influenzae and B. subtilis react to nutritional limitations when inducing competence (Redfield, 1993a,b). Notably, this is not the case for S. pneumoniae. In this species, nutrient starvation has never been observed to induce competence (Claverys et al., 2006); instead, during growth in rich medium the bacteria acquire competence in early log phase and maintain it only over a short period of time or differentially said ‘competence is induced in times of feast rather than famine’ (Claverys & Havarstein, 2007). (3) Neither H. influenzae nor B. subtilis induce competence upon DNA damage. (4) Unrelated DNA can be used as a nutrient source, especially because it could not recombine homologously into the chromosome. (5) The addition of ribonucleotide monophosphates, most notably AMP and GMP, to competence-inducing starvation medium reduced the natural transformation in H. influenzae by more than two orders of magnitude and significantly reduced the expression of competence genes in this organism (MacFadyen et al., 2001). These authors argued that the depletion of purines within the cell induces competence and that the incoming DNA could subsequently replenish the purine pool. Interestingly, this effect was not observable for desoxyribonucleotide monophosphates, triphosphates, or the free bases (MacFadyen et al., 2001). (6) The poor-quality DNA derived from dead cells might not be suitable for transformation-mediated evolution (Redfield, 1988; Redfield et al., 1997). But certain competent bacteria kill their (non-or not yet competent) siblings within the population, whereas other bacteria actively donate DNA through a T4SS (Hamilton et al., 2005; Hamilton & Dillard, 2006) or through a currently unknown mechanism (Stewart et al., 1983), suggesting that not all transforming DNA is of poor quality.
Recent studies on E. coli support Redfield's work. Finkel & Kolter (2001) provided evidence that E. coli can grow with DNA as the sole source of carbon and energy; however, this ability was disrupted in E. coli mutants lacking genes that encode for potential competence-related proteins. The authors of this and a follow-up study identified eight genes encoding proteins that are between 12% and 74% identical to the H. influenzae counterparts involved in natural competence (Finkel & Kolter, 2001; Palchevskiy & Finkel, 2006). In the absence of these genes, most of these strains encountered a stationary phase competition defect during co-culture with the parental wild-type bacteria. The authors of this study concluded that taking up DNA for nutritional purposes, ‘particularly when that DNA is heterologous and less likely to recombine onto the chromosome’ (Finkel & Kolter, 2001; Palchevskiy & Finkel, 2006), might confer a significant advantage even over the acquisition of a beneficial gene by HGT. However, because most of the homologous proteins identified in E. coli and other proteobacteria (Palchevskiy & Finkel, 2006) resemble the type IV pilus part of the DNA-uptake machinery, the question arises as to whether such DNA indeed reaches the cytoplasm as linear ssDNA, as occurs in naturally competent bacteria. Alternatively, the type IV pilus-like structure may assist in recruiting free dsDNA into the periplasm and thus facilitate transport across the outer membrane through the secretin PilQ/HofQ. DNA might be further degraded in the periplasm into nucleosides, which are subsequently taken up into the cytoplasm by specific nucleoside transporters to serve as a source of carbon and energy. Thus, whereas the first part of this process would resemble natural competence-induced DNA uptake and involve type IV pilus-like protein components, DNA transport across the inner membrane with concomitant degradation of one strand might not be identical in ‘nutritional competence’ (Palchevskiy & Finkel, 2006). Indeed, DNA uptake is a 2-step process in naturally competent H. pylori (Stingl et al., 2010); however, as discussed earlier, H. pylori uses a T4SS, not a type IV pilus-like structure, to shuffle the DNA across the outer membrane. But there are good indications that this 2-step DNA-uptake process also holds true for other Gram-negative bacteria, as demonstrated in a recent review (Krüger & Stingl, 2011). Furthermore, V. cholerae regulates the competence genes required for DNA movement across the outer membrane differentially from the competence genes whose products are involved in DNA translocation across the periplasmic space and the inner membrane (Lo Scrudato & Blokesch, 2012).
Other findings also oppose the ‘DNA for food’ hypothesis. For example, David Dubnau stated in a review from 1999 that B. subtilis possesses a powerful extracellular nuclease and adequate uptake systems for DNA degradation products (Dubnau, 1999). Extracellular nucleases have also been described for Vibrio cholerae (Newland et al., 1985; Focareta & Manning, 1987). Most notably, the main nuclease in this organism, Dns, is oppositely regulated from the competence genes that are directly involved in DNA uptake (Blokesch & Schoolnik, 2008; Lo Scrudato & Blokesch, 2012), and Dns is a major inhibitor of natural transformation in V. cholerae because it degrades the transforming material around the cell (Blokesch & Schoolnik, 2008). A role of this nuclease in the utilization of DNA as a nutrient source has been suggested (Blokesch & Schoolnik, 2008) and was experimentally supported with respect to the utilization of DNA as a phosphate source (Seper et al., 2011). Interestingly, the crystal structure of a concentrative nucleoside transporter of V. cholerae (NupC) has recently been solved. This protein uses a sodium-ion gradient for nucleoside transport across the inner membrane (Johnson et al., 2012) and may be involved in the uptake of nucleotides released by the extracellular nuclease Dns.
Another important point that undermines the DNA for food hypothesis is the energy associated with DNA uptake itself. As mentioned earlier and discussed in detail elsewhere (Chen & Dubnau, 2004; Allemand & Maier, 2009; Burton & Dubnau, 2010; Allemand et al., 2012), the DNA-uptake machinery is most likely a multiprotein complex (Box 1). Although the composition and mode of action of this complex has not fully been elucidated, the complex is assumed to resemble type IV pili (with the exception of H. pylori, as discussed earlier). Such type IV pilus-like structures (or shortened forms, also known as pseudopili; Pugsley, 1993; Chen & Dubnau, 2004) allow for the transport of DNA across the peptidoglycan layer and/or the outer membrane of Gram-positive and Gram-negative bacteria, respectively. An inner membrane channel subsequently allows translocation of the DNA across the inner membrane; this structure is probably conserved across all naturally transformable bacteria (Draskovic & Dubnau, 2005; Stingl et al., 2010; Suckow et al., 2011). Consistent with the resemblance of the components, the forces generated by type IV pilus retraction and DNA uptake were in the same range for both systems and represent some of the strongest linear motors characterized to date (Merz et al., 2000; Maier et al., 2002, 2004). As recently reviewed by Berenike Maier and others (Maier, 2005; Allemand & Maier, 2009; Allemand et al., 2012) such ‘directed DNA translocation is often energetically unfavourable and requires an active process that uses energy, namely the action of molecular motors’ (Allemand et al., 2012). Thus, the question arises as to whether the use of transforming DNA as an energy source would be able to supply enough energy to compensate for the consuming uptake process and still provide sufficient extra energy to be more cost-effective than the de novo synthesis of nucleotides. Furthermore, incoming ssDNA is protected against degradation in naturally competent bacteria by competence-specific proteins such as DprA (Mortier-Barriere et al., 2007). This mechanism is also more consistent with a role of the transforming DNA in DNA-repair processes or in the donation of new alleles/genes.
DNA for repair
Arguments for the repair hypothesis are principally based on the following facts:
The ability to take up DNA in some Gram-negative, naturally transformable bacteria is highly biased towards genetic material from the same or closely related species. Various strategies have evolved to this end (Fig. 2). For example, N. gonorrhoeae and H. influenzae discriminate between self and foreign DNA through the recognition of DUS that are overrepresented in their own genomes (Danner et al., 1980; Fitzmaurice et al., 1984; Elkins et al., 1991). Vibrio cholerae, in contrast, does not discriminate between self and foreign DNA at the level of the DNA uptake (Suckow et al., 2011). Because competence induction in this organism is tightly linked with an accumulation of the species-specific autoinducer CAI-1 (Suckow et al., 2011; Lo Scrudato & Blokesch, 2012), species-specific DNA is highly likely to reach the cytosol. In contrast, H. pylori does not display any preference for species-specific DNA. This assumption is based on the fact that competence is constitutive in this organism and that DUS-dependent sorting does not occur at the level of the DNA-uptake machinery. However, recent data has indicated that a mechanism based on R-M systems might control DNA uptake in H. pylori, as explained above (Aras et al., 2002; Humbert & Salama, 2008; Humbert et al., 2011). This system may also ensure the species-specificity of transforming DNA by recognizing and degrading foreign genetic material, and protecting the genome from foreign DNA (Fig. 2; Aras et al., 2002; Humbert & Salama, 2008; Humbert et al., 2011). Another source for species-specific DNA may be fratricide. More precisely, bacterial fratricide is associated with natural competence of S. pneumoniae (Guiral et al., 2005; Havarstein et al., 2006 and recent review by Claverys & Havarstein, 2007) and probably also in H. pylori (Dorer et al., 2010; Fig. 2). In this context, bacterial cells of the same population are killed to provide transforming DNA. Based on several examples of the intentional killing of bacterial siblings, Gilmore & Haas (2005) concluded that ‘the selective lysis of siblings by a subpopulation of bacterial cells appears to be a highly evolved and complex process’.
Based on these important points, the idea that natural transformation serves as a mechanism of DNA repair seems sound. In this scenario, the uptake of DNA from closely related organisms would facilitate the maintenance of genomic integrity. Evidence from experiments on the naturally competent Gram-positive bacterium B. subtilis supports this hypothesis (Michod et al., 1988; Wojciechowski et al., 1989), but recent findings on the two Gram-negative bacteria, H. pylori and L. pneumophila, with nonsense mutations in their DNA-uptake systems, did not support the repair hypothesis (Dorer et al., 2010; Charpentier et al., 2011). In these studies the bacterial strains incapable of DNA uptake showed no increased sensitivity to genotoxic agents (Dorer et al., 2010; Charpentier et al., 2011).
DNA for evolution
Finally, natural transformation might enable rapid evolution when diversity may be beneficial; these circumstances include such stresses as high population densities, DNA damage, abundance or a lack of certain carbon sources and/or starvation. All of these conditions are known to induce natural competence in at least a subset of naturally transformable bacteria, as described earlier and illustrated in Fig. 1. A recent study on the long-term in vitro passage of H. pylori (c. 1000 generations) supported an evolutionary advantage of natural transformation because competent bacteria increased their fitness faster than those unable to take up external DNA (Baltrus et al., 2008). Another recent study investigated the occurrence of multi-drug-resistant (MDR) strains based on recombination following DNA uptake as part of the natural transformation process of A. baylyi (Perron et al., 2012). The authors demonstrated that in the presence of recombination, resistance genes were readily exchanged, and MDR strains were obtained within fewer generations (Perron et al., 2012).
However, as noted by Perron et al. and as described in depth in a recent review on this topic (MacLean et al., 2010), it is important that ‘evolution experiments offer a useful approach to uncover the factors determining the evolution of resistance, but most experiments have studied clonal populations without any contribution of recombination’. Furthermore, the ‘benefits of recombination are context-dependent’, and experimental setups are crucial to the outcome of such experiments. Such context dependency was also highlighted in a recent study by Engelmoer & Rozen (2011). These authors emphasized once more the biased setup of most experimental studies, which only examine benefits dependent on the acquisition of DNA as part of natural transformation. However, because natural competence is a developmental programme and often induces other processes apart from DNA uptake (e.g. fratricide in S. pneumoniae or competence-dependent growth arrest, as seen in B. subtilis), benefits arising from these processes might have been overlooked in the past (Engelmoer & Rozen, 2011). In this study, the authors investigated the natural competence of S. pneumoniae and confirmed that transformation is beneficial in a ‘DNA for repair’ scenario upon treating cells with DNA-damaging agents (Engelmoer & Rozen, 2011), but they also provided evidence that competence is beneficial in withstanding other kinds of stresses and that these benefits do not rely on transformation (Engelmoer & Rozen, 2011). The authors concluded that their findings were in line with ‘Claverys’ hypothesis (Claverys et al., 2006) that competence but not necessarily transformation may act as a general process to relieve stress’ (Engelmoer & Rozen, 2011).
In summary, we conclude that there are many benefits of natural competence and transformation, and there might be no single reason that transformation is maintained in bacteria. The fact that numerous bacteria are known or predicted to be naturally transformable is a strong indication of the importance of this mode of HGT.