Competence, DNA Uptake and Transformation in Pasteurellaceae

e ability to take up DNA from the environment and recombine it into the chromosome appears to be ancestral to the Pasteurellaceae, although only some isolates do this efficiently under laboratory conditions. Studies of readily transformable isolates have shown that competence for DNA uptake is regulated by the cyclic AMP-dependent regulatory protein CRP and by Sxy, a competence-specific transcriptional activator. Once cells are competent, DNA uptake is promoted by recognition of an uptake signal sequence motif that is highly over-represented in the genomes of all Pasteurellaceae, including those that cannot be transformed. Transport of the DNA across the cell envelope uses components of the type 4 pilus machinery, homologous to those used by other naturally competent bacteria. Once in the cytoplasm this DNA may be degraded or, if sequence similarity permits, it may be recombined into the chromosome. Although such recombination can have important evolutionary consequences, DNA uptake is likely to serve primarily as a source of nucleotides for the cell. Introduction !e family Pasteurellaceae has historically drawn much attention because of its many pathogenic members. However, another trait described in several of its members has also attracted the interest of biologists from fields including pathogenesis and molecular and evolutionary biology. !is is natural competence, the ability of intact, living cells to actively take up DNA from their extracellular environment. If the sequences are sufficiently similar, this DNA may recombine with the host genome. When such a recombination event changes the cell’s genotype, the cell is said to be transformed. It is important to begin by clarifying the difference between natural competence and two other phenomena, artificially induced competence and transformation. Natural competence differs from artificially induced competence, where cells are chemically treated or exposed to high electrical currents to allow entry of DNA into the cell. Second, transformation is not a necessary consequence of DNA uptake, both because much of the DNA taken up by competent cells is degraded and because cells that take up unrelated or genetically identical DNAs will not be transformed. In this chapter, we first give a brief overview of competence development, DNA uptake and transformation. We then provide detailed reviews of each of these topics and consider the evolutionary implications of competence. We then conclude with suggestions for improving transformability of problematic strains. Historically, Haemophilus influenzae is the best-studied Pasteurellaceae species with respect to competence; unless otherwise stated, gene names and numbers in this chapter refer to the sequenced H. influenzae Rd strain. Like most other bacteria, Pasteurellaceae species develop competence only under certain conditions (i.e. competence is not constitutive). In H. influenzae, competence is induced under conditions of depleted carbon and energy sources Competence, DNA Uptake and Transformation in Pasteurellaceae Heather Maughan, Sunita Sinha, Lindsay Wilson and Rosemary Redfield 4 UNCORRECTED FIRST PROOFS Maughan et al. 82 | (Macfadyen et al., 1996). Competence induction causes an initial increase in cAMP (cofactor of the cAMP receptor protein CRP, also known as CAP). Active CRP first causes increased transcription of the gene encoding the competence activator protein Sxy. CRP and Sxy together then induce the expression of the genes in the competence regulon. !e products of many of these genes play essential roles in DNA uptake, with proteins homologous to components of the type IV pilus machinery (Tfp) thought to be responsible for binding DNA and pulling it across the outer membrane into the periplasm. DNA normally crosses the outer membrane in its double-stranded form, but one strand is then degraded during translocation across the inner membrane into the cytoplasm; the nucleotides released are rapidly reused for new DNA synthesis (Goodgal, 1982). If sequence similarity permits, the other strand may recombine with a homologous sequence in the chromosome; otherwise it too will be degraded. Although much of this machinery appears to be shared by other bacteria, one aspect of DNA uptake in Pasteurellaceae is shared only with the Neisseriaceae: they do not indiscriminately take up external DNA, but instead show a strong bias for DNA fragments containing specific uptake signal sequence (USS) motifs. !e motifs of the two families are unrelated. Because USSs are present at more than thousand sites in Pasteurellaceae genomes, the bias leads to preferential uptake of DNA derived from relatives. Competence and transformation have medical relevance at several levels, because antibiotic resistance genes, virulence determinants and capsular serotype genes are spread by transformation (Kroll and Moxon, 1990; Kroll et al., 1998; Maiden, 1998). Tfp genes are specifically induced upon host cell contact by Actinobacillus pleuropneumoniae (Boekema et al., 2004) and during biofilm formation by H .influenzae (Bakaletz et al., 2005; Jurcisek and Bakaletz, 2007). !e DNA abundantly present in respiratory mucus may therefore be an important nutrient for these bacteria (Lethem et al., 1990). It follows that, in order to understand how Pasteurellaceae pathogens exploit their specific environments, we must understand the regulation and mechanism of competence. Distribution of competence in Pasteurellaceae In the laboratory, the most sensitive way to detect natural competence is to measure transformation by genetically marked donor DNA; in the Pasteurellaceae this is most conveniently done with DNA carrying an antibiotic resistance gene. !ese assays express competence as a ‘transformation frequency’, the ratio of bacteria that have recombined the marked DNA to the total bacteria present. Competence can also be directly assessed by measuring the uptake of radiolabelled donor DNA, but this is less sensitive by several orders of magnitude. Transformation has been observed in laboratory cultures of at least some isolates of three of the eight sequenced Pasteurellaceae species: H. influenzae (Alexander and Leidy, 1951), Aggregatibacter actinomycetemcomitans (Wang et al., 2002) and A. pleuropneumoniae (Bosse et al., 2004). [Haemophilus] parasuis (Bigas et al., 2005) and Haemophilus parainfluenzae (Nickel and Goodgal, 1964) are also transformable. Pasteurella multocida has not been demonstrated to be competent, but its genome sequence (May et al., 2001) contains all of the genes known to be necessary for competence development in H. influenzae. Homologues of H. influenzae competence genes in the sequenced Pasteurellaceae genomes are listed in Table 4.1, with footnotes indicating genes with defects likely to preclude function. All of the genes known to be needed for DNA uptake by H. influenzae are present in all of the other genomes, although one or more are obviously defective in [Haemophilus] ducreyi, Histophilus somni, Mannheimia haemolytica and ‘Mannheimia succiniciproducens’ (Challacombe et al., 2007; Gioia et al., 2006; Hong et al., 2004). !ese strains (though perhaps not all strains of these species) are thus unlikely to be able to develop full competence under any conditions. Different strains of Pasteurellaceae species are known to exhibit very different transformation frequencies, as do different strains in other bacterial families. Some strains are completely nontransformable in the laboratory. Transformability varied over three orders of magnitude in different UNCORRECTED FIRST PROOFS Ta bl e 4. 1 O rt ho lo gu es o f H . i nfl ue nz ae R d K W 20 g en es in th e C R P -S re gu lo n N am e H ae m op hi lu s