Evolution of Natural Transformation: Testing the DNA Repair Hypothesis in Bacillus Subtilis and Haemophilus Influenzae

The hypothesis that the primary function of bacterial transformation is DNA repair was tested in the naturally transformable bacteria Bacillus subtilis and Haemophilus influenzae by determining whether competence for transformation is regulated by DNA damage. Accordingly, DNA damage was induced by mitomycin C and by ultraviolet radiation at doses that efficiently induced a known damage-inducible gene fusion, and the ability of the damaged cultures to transform was monitored. Experiments were carried out both under conditions where cells do not normally become competent and under competence-inducing conditions. No induction or enhancement of competence by damage was seen in either organism. These experiments strongly suggest that the regulation of competence does not involve a response to DNA damage, and thus that explanations other than DNA repair must be sought for the evolutionary functions of natural transformation systems.     

Natural transformation of Campylobacter jejuni requires components of a type II secretion system

The human pathogen Campylobacter jejuni is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes. However, in C. jejuni little is known about the genes involved in this process. We used random transposon mutagenesis to identify genes that are required for the transformation of this organism. We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane. Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species. Other genes identified in our screen encode proteins unique to C. jejuni or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria.     

Natural DNA uptake by Escherichia coli

Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.     

Episodic Selection and the Maintenance of Competence and Natural Transformation in Bacillus subtilis

We present a new hypothesis for the selective pressures responsible for maintaining natural competence and transformation. Our hypothesis is based in part on the observation that in Bacillus subtilis, where transformation is widespread, competence is associated with periods of nongrowth in otherwise growing populations. As postulated for the phenomenon of persistence, the short-term fitness cost associated with the production of transiently nongrowing bacteria can be compensated for and the capacity to produce these competent cells can be favored due to episodes where the population encounters conditions that kill dividing bacteria. With the aid of a mathematical model, we demonstrate that under realistic conditions this “episodic selection” for transiently nongrowing (persisting) bacteria can maintain competence for the uptake and expression of exogenous DNA transformation. We also show that these conditions for maintaining competence are dramatically augmented even by rare episodes where selection favors transformants. Using experimental populations of B. subtilis and antibiotic-mediated episodic selection, we test and provide support for the validity of the assumptions behind this model and the predictions generated from our analysis of its properties. We discuss the potential generality of episodic selection for the maintenance of competence in other naturally transforming species of bacteria and critically evaluate other hypotheses for the maintenance (and evolution) of competence and their relationship to this hypothesis.     

DNA Repair and the Evolution of Transformation in Bacillus Subtilis. II. Role of Inducible Repair

In Bacillus subtilis, DNA repair and recombination are intimately associated with competence, the physiological state in which the bacterium can bind, take up and recombine exogenous DNA. Previously, we have shown that the homologous DNA transformation rate (ratio of transformants to total cells) increases with increasing UV dosage if cells are transformed after exposure to UV radiation (UV-DNA), whereas the transformation rate decreases if cells are transformed before exposure to UV (DNA-UV). In this report, by using different DNA repair-deficient mutants, we show that the greater increase in transformation rate in UV-DNA experiments than in DNA-UV experiments does not depend upon excision repair or inducible SOS-like repair, although certain quantitative aspects of the response do depend upon these repair systems. We also show that there is no increase in the transformation rate in a UV-DNA experiment when repair and recombination proficient cells are transformed with nonhomologous plasmid DNA, although the results in a DNA-UV experiment are essentially unchanged by using plasmid DNA. We have used din operon fusions as a sensitive means of assaying for the expression of genes under the control of the SOS-like regulon in both competent and noncompetent cell subpopulations as a consequence of competence development and our subsequent experimental treatments. Results indicate that the SOS-like system is induced in both competent and noncompetent subpopulations in our treatments and so should not be a major factor in the differential response in transformation rate observed in UV-DNA and DNA-UV treatments. These results provide further support to the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in the repair of DNA damage.     

Competence-induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during cocultivation

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.     

DNA Repair and the Evolution of Transformation in Haemophilus Influenzae

Under certain environmental conditions, naturally transforming bacteria are induced to pick up DNA released into the environment by other cells of the same or closely related species and, by homologous recombination, integrate that DNA into their chromosome. The selective pressures responsible for the evolution and maintenance of this form of genetic outcrossing, or sex, in bacteria are not known. A prominent hypothesis is that transformation, and sex in general, evolved as a means of obtaining DNA templates to repair damaged regions of the chromosome. Previous results obtained with Bacillus subtilis were consistent with the repair hypothesis. In an effort to explore the generality of those results, I have tested the repair hypothesis with Haemophilus influenzae, a naturally transforming, gram-negative species of bacteria. The results of UV damage-survivorship experiments with H. influenzae were also consistent with that hypothesis. However, additional experiments demonstrate that the higher survival of transformed cultures cannot be accounted for by use of the transforming DNA as templates for repair. I consider alternative hypotheses for the means by which transformation can increase cell survival following UV exposure and discuss the implications of these results with respect to the DNA repair hypothesis and the evolution of transformation.     

Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili

We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).     

Could DNA uptake be a side effect of bacterial adhesion and twitching motility?

DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence—a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.     

Deleterious effects of recombination and possible nonrecombinatorial advantages of sex in a fungal model

Why sexual reproduction is so prevalent in nature remains a major question in evolutionary biology. Most of the proposed advantages of sex rely on the benefits obtained from recombination. However, it is still unclear whether the conditions under which these recombinatorial benefits would be sufficient to maintain sex in the short term are met in nature. Our study addresses a largely overlooked hypothesis, proposing that sex could be maintained in the short term by advantages due to functions linked with sex, but not related to recombination. These advantages would be so essential that sex could not be lost in the short term. Here, we used the fungus Aspergillus nidulans to experimentally test predictions of this hypothesis. Specifically, we were interested in (i) the short‐term deleterious effects of recombination, (ii) possible nonrecombinatorial advantages of sex particularly through the elimination of mutations and (iii) the outcrossing rate under choice conditions in a haploid fungus able to reproduce by both outcrossing and haploid selfing. Our results were consistent with our hypotheses: we found that (i) recombination can be strongly deleterious in the short term, (ii) sexual reproduction between individuals derived from the same clonal lineage provided nonrecombinatorial advantages, likely through a selection arena mechanism, and (iii) under choice conditions, outcrossing occurs in a homothallic species, although at low rates.     

Optimal Strategy for Competence Differentiation in Bacteria

A phylogenetically diverse subset of bacterial species are naturally competent for transformation by DNA. Transformation entails recombination of genes between different lineages, representing a form of bacterial sex that increases standing genetic variation. We first assess whether homologous recombination by transformation is favored by evolution. Using stochastic population genetic computer simulations in which beneficial and deleterious mutations occur at many loci throughout the whole genome, we find that transformation can increase both the rate of adaptive evolution and the equilibrium level of fitness. Secondly, motivated by experimental observations of Bacillus subtilis, we assume that competence additionally entails a weak persister phenotype, i.e., the rates of birth and death are reduced for these cells. Consequently, persisters evolve more slowly than non-persisters. We show via simulation that strains which stochastically switch into and out of the competent phenotype are evolutionarily favored over strains that express only a single phenotype. Our model''s simplicity enables us to derive and numerically solve a system of finite- deterministic equations that describe the evolutionary dynamics. The observed tradeoff between the benefit of recombination and the cost of persistence may explain the previously mysterious observation that only a fractional subpopulation of B. subtilis cells express competence. More generally, this work demonstrates that population genetic forces can give rise to phenotypic diversity even in an unchanging and homogeneous environment.     

comH, a novel gene essential for natural transformation of Helicobacter pylori

Helicobacter pylori is naturally competent for transformation, but the DNA uptake system of this bacterium is only partially characterized, and nothing is known about the regulation of competence in H. pylori. To identify other components involved in transformation or competence regulation in this species, we screened a mutant library for competence-deficient mutants. This resulted in the identification of a novel, Helicobacter-specific competence gene (comH) whose function is essential for transformation of H. pylori with chromosomal DNA fragments as well as with plasmids. Complementation of comH mutants in trans completely restored competence. Unlike other transformation genes of H. pylori, comH does not belong to a known family of orthologous genes. Moreover, no significant homologs of comH were identified in currently available databases of bacterial genome sequences. The comH gene codes for a protein with an N-terminal leader sequence and is present in both highly competent and less-efficient transforming H. pylori strains. A comH homolog was found in Helicobacter acinonychis but not in Helicobacter felis and Helicobacter mustelae.     

EXTENSIVE VARIATION IN NATURAL COMPETENCE IN HAEMOPHILUS INFLUENZAE

The ability of some bacteria to take up and recombine DNA from the environment is an important evolutionary problem because its function is controversial; although populations may benefit in the long-term from the introduction of new alleles, cells also reap immediate benefits from the contribution of DNA to metabolism. To clarify how selection has acted, we have characterized competence in natural isolates of H. influenzae by measuring DNA uptake and transformation. Most of the 34 strains we tested became competent, but the amounts of DNA they took up and recombined varied more than 1000-fold. Differences in recombination were not due to sequence divergence and were only partly explained by differences in the amounts of DNA taken up. One strain was highly competent during log phase growth, unlike the reference strain Rd, but several strains did not develop competence under any of the tested conditions. Analysis of competence genes identified genetic defects in two poorly transformable strains. These results show that strains can differ considerably in the amount of DNA they take up and recombine, indicating that the benefit associated with competence is likely to vary in space and/or time.     

Regulation of hypercompetence in Legionella pneumophila

Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable. Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake. We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction. For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37 degrees C. However, it was previously reported that microaerophilic growth at 37 degrees C allows L. pneumophila serogroup 1 strain AA100 to be naturally transformed. Here we report that another L. pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions. Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30 degrees C. In contrast to Lp02, AA100 is only minimally transformable at 30 degrees C, indicating that Lp02 is hypercompetent under these conditions. To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake. Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L. pneumophila. This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable.     

The availability of purine nucleotides regulates natural competence by controlling translation of the competence activator Sxy

Many bacteria are naturally competent, able to bind and take up DNA from their extracellular environment. This DNA can serve as a significant source of nutrients, in addition to providing genetic material for recombination. The regulation of competence in several model organisms highlights the importance of this nutritional function, although it has often been overlooked. Natural competence is induced by starvation in Haemophilus influenzae, the model for competence regulation in the gamma‐proteobacteria. This induction depends on the activation of the global metabolic regulator CRP, which occurs upon depletion of phosphotransferase sugars. In this work, we show that the depletion of purine nucleotides under competence‐inducing conditions activates the CRP‐dependent competence‐specific regulator Sxy. Depletion of extra‐ or intra‐cellular purine nucleotides activates Sxy translation, while high levels inhibit it. This is modulated by the stem structure formed by sxy mRNA. The exact mechanism by which the nucleotide depletion signal is transduced is unclear, but it does not involve direct binding of purine intermediates to the sxy stem, and does not require Hfq or competence proteins. Similar regulation occurs in the relatives of H. influenzae, Actinobacillus pneumoniae and A. suis, confirming the importance of processes enabling competent bacteria to exploit the abundant DNA in their environments.     

Functional characterization of the competence protein DprA/Smf in Escherichia coli

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.     

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.     

Properties and biological role of streptococcal fratricins

Competence for natural genetic transformation is widespread in the genus Streptococcus. The current view is that all streptococcal species possess this property. In addition to the proteins required for DNA uptake and recombination, competent streptococci secrete muralytic enzymes termed fratricins. Since the synthesis and secretion of these cell wall-degrading enzymes are always coupled to competence development in streptococci, fratricins are believed to carry out an important function associated with natural transformation. This minireview summarizes what is known about the properties of fratricins and discusses their possible biological roles in streptococcal transformation.     

Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.