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Evolution of competence and DNA uptake specificity in the Pasteurellaceae Redfield, Rosemary J; Findlay, Wendy A; Bossé, Janine; Kroll, J S; Cameron, Andrew D; Nash, John H Oct 12, 2006

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ralssBioMed CentBMC Evolutionary BiologyOpen AcceResearch articleEvolution of competence and DNA uptake specificity in the PasteurellaceaeRosemary J Redfield*1, Wendy A Findlay2, Janine Bossé3, J Simon Kroll3, Andrew DS Cameron4 and John HE Nash2Address: 1Dept. of Zoology, University of British Columbia, Vancouver BC Canada, 2Institute for Biological Sciences, National Research Council of Canada, Ottawa ON Canada, 3Dept. of Paediatrics, Faculty of Medicine, Imperial College London, London W2 1PG UK and 4Dept. of Microbiology and Immunology, University of British Columbia, Vancouver BC CanadaEmail: Rosemary J Redfield* - redfield@zoology.ubc.ca; Wendy A Findlay - Wendy.Findlay@nrc-cnrc.gc.ca; Janine Bossé - j.bosse@imperial.ac.uk; J Simon Kroll - s.kroll@imperial.ac.uk; Andrew DS Cameron - dafydd@interchange.ubc.ca; John HE Nash - John.Nash@nrc-cnrc.gc.ca* Corresponding author    AbstractBackground: Many bacteria can take up DNA, but the evolutionary history and function of natural competenceand transformation remain obscure. The sporadic distribution of competence suggests it is frequently lost and/orgained, but this has not been examined in an explicitly phylogenetic context. Additional insight may come fromthe sequence specificity of uptake by species such as Haemophilus influenzae, where a 9 bp uptake signal sequence(USS) repeat is both highly overrepresented in the genome and needed for efficient DNA uptake. We used thedistribution of competence genes and DNA uptake specificity in H. influenzae's family, the Pasteurellaceae, toexamine the ancestry of competence.Results: A phylogeny of the Pasteurellaceae based on 12 protein coding genes from species with sequencedgenomes shows two strongly supported subclades: the Hin subclade (H. influenzae, Actinobacillusactinomycetemcomitans, Pasteurella multocida, Mannheimia succiniciproducens, and H. somnus), and the Apl subclade(A. pleuropneumoniae, M. haemolytica, and H. ducreyi). All species contained homologues of all known H. influenzaecompetence genes, consistent with an ancestral origin of competence. Competence gene defects were identifiedin three species (H. somnus, H. ducreyi and M. haemolytica); each appeared to be of recent origin.The assumption that USS arise by mutation rather than copying was first confirmed using alignments of H.influenzae proteins with distant homologues. Abundant USS-like repeats were found in all eight Pasteurellaceangenomes; the repeat consensuses of species in the Hin subclade were identical to that of H. influenzae(AAGTGCGGT), whereas members of the Apl subclade shared the consensus ACAAGCGGT. All species' USSshad the strong consensus and flanking AT-rich repeats of H. influenzae USSs. DNA uptake and competitionexperiments demonstrated that the Apl-type repeat is a true USS distinct from the Hin-type USS: A.pleuropneumoniae preferentially takes up DNA fragments containing the Apl-type USS over both H. influenzae andunrelated DNAs, and H. influenzae prefers its own USS over the Apl type.Conclusion: Competence and DNA uptake specificity are ancestral properties of the Pasteurellaceae, withdivergent USSs and uptake specificity distinguishing only the two major subclades. The conservation of mostcompetence genes over the ~350 million year history of the family suggests that lineages that lose competencePublished: 12 October 2006BMC Evolutionary Biology 2006, 6:82 doi:10.1186/1471-2148-6-82Received: 12 June 2006Accepted: 12 October 2006This article is available from: http://www.biomedcentral.com/1471-2148/6/82© 2006 Redfield et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 15(page number not for citation purposes)may be evolutionary dead ends.BMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82BackgroundMany bacteria are able to take up DNA from the environ-ment [1]. DNA provides these naturally competent cellswith nutrients (nucleotides, N and P), while recombina-tion of incoming DNA with the cell's genome can alsoprovide new genetic information. However, many aspectsof the evolution of competence remain unclear.Competence is widely distributed among bacteria, andsome of the genes required for DNA uptake are sharedbetween even distant relatives, suggesting an ancient com-mon origin for competence. For example, the Gram posi-tive bacteria Bacillus subtilis and Streptococcus pneumoniaeand the Gram negative Neisseria gonorrhoeae and Haemo-philus influenzae all require homologues of type four pilusproteins and of the ComEC/Rec2 membrane channel [1].However, the regulatory processes controlling expressionof these competence genes are very different in the differ-ent organisms [2]. Furthermore the distribution of naturalcompetence is surprisingly sporadic; most naturally com-petent bacteria have many relatives, including otherstrains of the same species, that cannot be transformedunder laboratory conditions (for examples see [3-6]). Twoexplanations seem equally plausible. First, competencemight be ancestral to most major lineages but frequentlylost (and possibly regained, under different regulation).Alternatively, competence might be frequently gained inindependent lineages, e.g. if the genetic requirements forDNA uptake are simple and readily met by laterally trans-ferred genes or by mutation of genes with related func-tions such as those associated with type IV pili.The uptake specificity of some naturally competent bacte-ria can also guide inferences about the evolution of com-petence. Although many naturally competent bacteria willtake up DNA fragments from any source with equal effi-ciency, members of some Gram-negative families take upDNA fragments from their own species much more effi-ciently than unrelated DNA. In the Pasteurellaceae andNeisseriaceae the molecular basis of this specificity is pref-erential binding of the uptake machinery to short DNAsequences present in thousands of copies in each species'genome. Such sequences are referred to as uptake signalsequences (USSs) in the Pasteurellaceae and DNA uptakesequences (DUSs) in the Neisseriaceae; they are not knownin other naturally transformable bacteria [7,8].The best-characterized uptake sequences are those of Hae-mophilus influenzae and Neisseria meningitidis and N. gonor-rhoeae. The preferred sequences themselves appear to havelittle in common: the core H. influenzae USS is 5'-AAGT-GCGGT (5'-ACCGCACTT in the reverse orientation), withtwo AT-rich motifs on the 3' side of the standard orienta-frequencies and distributions suggest that they have arisenby similar processes. Both USSs and DUSs are present intheir respective genomes at frequencies close to one copyper kb and both show no significant orientation bias.Both types are distributed somewhat more regularlyaround their genomes than expected for randomly locatedrepeats, but both have some copies occurring in closelyspaced oppositely oriented pairs [7]. Both USSs and DUSsare preferentially found in non-coding DNA sequences,but both have many copies in coding sequences. Bothtypes are also overrepresented in the genomes of at leastsome other members of their genus or family [4,7-9,11-13].One puzzling attribute shared by USSs and DUSs is anunusually strong consensus, with each genome contain-ing many more copies that perfectly match its consensuscore sequence than singly mismatched copies. This pat-tern is typical of young transposons and other genetic ele-ments that multiply by copying, but very different fromthe more relaxed consensus typical of sequences that func-tion as binding sites for regulatory proteins, which ariseby point mutation of pre-existing sequences. BecauseUSSs are thought to function by binding to DNA-receptorproteins at the cell surface [14,15], their very strong con-sensus is anomalous. The explanation might be that theDNA uptake machinery at the cell surface binds DNA withmuch higher specificity than do intracellular DNA-bind-ing proteins. However the possibility that USSs arise by acopying process has not been excluded.Previous analysis has found that copies of the H. influen-zae USS are abundant in the genomes of several othermembers of the Pasteurellaceae (Actinobacillus actinomyce-temcomitans, Pasteurella multocida, and H. somnus), andcomparison of homologous genes in H. influenzae and P.multocida has shown that individual USSs can be stableover hundreds of millions of years [13]. However, prelim-inary examinations of the sequenced genomes of the Pas-teurellaceans Mannheimia haemolytica (by SarahHighlander) and Actinobacillus pleuropneumoniae (by our-selves) found that the H. influenzae USS was much lessabundant than a related sequence that differs at severalpositions, suggesting that these genomes might contain avariant USS.Insight into the evolution of competence will depend onan improved understanding of Pasteurellaceaen phylog-eny. Almost all Pasteurellaceae (gamma-proteobacteria)are commensals and/or pathogens of the mucosal surfacesof vertebrates, primarily birds and mammals, and severalare important human pathogens. Although phylogeneticanalysis based on 16S rRNA sequences has confirmed thatPage 2 of 15(page number not for citation purposes)tion [9], whereas the Neisseria DUS is GCCGTCTGAA withno flanking motifs [10]. However, similarities in genomicthe family is monophyletic, the relationships of its mem-bers remain poorly resolved. Two recently published phy-BMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82logenies used small-subunit rRNA sequences from 83Pasteurellacean taxa [16] and partial sequences of thehousekeeping genes atpD, infB and rpoB from 28–36strains [17], but the resolution was unsatisfactory, withmany low bootstrap values and unresolved nodes.Here we use the concatenated sequences of 12 proteins toconstruct a well-resolved phylogenetic tree for the speciesof Pasteurellaceae with genome sequences available. Thistree then serves as a framework against which we charac-terize the long-term evolution of competence genes and ofDNA uptake specificity.Results and DiscussionA robust Pasteurellacean phylogenyThe amino acid sequences of 12 well-conserved geneswere identified from the available published and draftsequences of Pasteurellacean genomes and used to inferthe consensus phylogeny shown in Figure 1. HomologousE. coli genes were used as the outgroup. The chosen genesdid not contain the H. influenzae USS, were distributedaround the H. influenzae genome, and had strong homo-logues in the other genomes. Intracellular proteins werechosen to preclude the diversifying selection that can biasevolution of proteins exposed on the cell surface, andgenes with base compositions typical of their species'genomes were used to preclude recent horizontal transfer.The resulting phylogenetic tree (Fig. 1) identified two pri-mary subclades within the Pasteurellaceae. These arereferred to below as the Hin subclade and the Apl subc-lade. The statistical support for these subclades is robust,with bootstrap values of 100%. Independent phylogeniessubclade from the five other species. The 100% bootstrapvalues place A. pleuropneumoniae and H. ducreyi as sisterspecies, but the branching order in the Hin subcladeremains uncertain.This phylogeny is restricted to the eight species withsequenced genomes, but it is the first Pasteurellacean treeto have strong statistical support. It differs in manyrespects from both the 16S rRNA and protein phylogeniespreviously published for the Pasteurellaceae. However inour view these discrepancies are the consequence of mostbranches of those earlier phylogenies having very poorbootstrap support, making them intrinsically unreliable,and so should not be a cause for concern. The Apl subcladeit predicts was also supported by the protein tree of Chris-tensen et al. [17]. Although the Apl subclade is not seen inChristensen et al.'s small-subunit rRNA tree, the resolu-tion of that region of their tree is poor (best bootstraps are69% and 75%) [16]. The topology of an earlier tree basedon small subunit rRNA sequences agrees with ours,although none of the relevant bootstrap values in that treeare significant [18]. The new tree also confirms what theprevious more-detailed but less-well-supported trees hadpredicted – that within the Pasteurellaceae true evolution-ary relatedness is not well correlated with many of the fea-tures previously used to assign isolates to genus[17,19,20].The genus assignment of M. succiniciproducens provides anexample. This species, isolated from bovine rumen, wasassigned to Mannheimia based on a simple small-subunitrRNA tree with no bootstrap analysis [21]. Our phyloge-netic analysis instead places the two Mannheimia speciesPhylogeny of 8 Pasteurellacean speciesFigure 1Phylogeny of 8 Pasteurellacean species. Phylogenetic analysis was based on amino acid sequence of 12 protein-coding genes (listed in Methods) with homologues in all 8 Pasteurellacean genomes and in E. coli. The scale bar is 0.1 substitutions per site.A. actinomycetemcomitansP. multocidaH. somnusM. succiniciproducensH. influenzaeH. ducreyiM. haemolyticaA. pleuropneumoniaeE. coli8096100100100800.1Page 3 of 15(page number not for citation purposes)done for each protein separately all grouped A. pleuropneu-moniae, M. haemolytica and H. ducreyi together as a distinctin separate subclades. The 80% bootstrap score support-ing M. succiniciproducens's placement as the sister group toBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82H. influenzae in Fig. 1 is too low to rule out a closer affinitywith P. multocida. Hong et al. compared the M. succinicip-roducens genome sequence to those of both P. multocidaand H. influenzae; more genes are shared with the former,but the amino acid identities are higher with the latter[22,23]. In any case it is striking to find a rumen bacteriumas such a close relative of bacteria otherwise restricted torespiratory mucosa.Competence genes in Pasteurellacean genomesNatural transformation has been demonstrated experi-mentally in only three of the eight sequenced species (H.influenzae, A. actinomycetemcomitans and A. pleuropneumo-niae [12,24,25]). Only two other species within the Pas-teurellaceae have also been shown to be naturallycompetent (Haemophilus parasuis [26] and Haemophilusparainfluenzae [4,27]). A number of other species haveresisted multiple attempts at transformation in the labora-tory, but their nontransformability could be misleading,as cellular processes important in the natural environ-ment may not be induced under laboratory culture condi-tions.VanWagoner et al. identified homologues of several H.influenzae competence genes (HI0366, HI0938 andHI0939) in most sequenced Pasteurellacean genomes[28]. As we have recently identified the complete compe-tence regulon of H. influenzae, we examined the genomesgenomes contain recognizable homologues of all of thegenes known to be required for competence in H. influen-zae, as well as homologues of most other genes consist-ently occurring in the same operons.However not all of the genes in this ancestor's sequenceddescendants appear to be functional. The H. ducreyi comA,comB and comM genes are interrupted by an internal stopcodon (comA) and frameshifts (comB and comM). A dele-tion in the H. somnus genome fuses the 5' portion of comDto the 3' 67% of comE, which also contains a frameshift. A17 kb insertion disrupts the comM gene of M. succinicipro-ducens, and most of pilB in M. haemolytica has beendeleted. In some cases, examination of genome sequencesfrom different isolates revealed discrepancies; these mayresult from strain-specific variation or from the prelimi-nary nature of some of the sequences used. Only thesequenced genomes of H influenzae, A. actinomycetemcom-itans, P. multocida and A. pleuropneumoniae retain fullyintact sets of competence genes.What inferences can be drawn about the evolution ofcompetence? First, the most parsimonious explanationfor the presence of all competence genes in all genomes isthat the ancestral Pasteurellacean had functional copies ofall these genes and was naturally competent. When andwhere did this ancestor live? Although dating bacterialdivergences is highly problematic, the most recent com-Table 1: Competence genes in Pasteurellacean genomesGene HI# SpeciesHin Aac Pmu Hso Msu Apl Mha HducomA 0439 + + + + + + + defcomB 0438 + + + + + + + defcomC 0437 + + + + + + + +comD 0436 + + + def + + + +comE 0435 + + + def + + + +comF 0434 + + + + + + + +comE1 1008 + + + + + + + +comM 1117 + + + + def + + defdprA 0985 + + + + + + + +pilA 0299 + + + + + + + +pilB 0298 + + + + + + def +pilC 0297 + + + + + + + +pilD 0296 + + + + + + + +rec2 0061 + + + + + + + +sxy 0601 + + + + + + + +0366 + + + + + + + +0938 + + + + + + + +0939 + + + + + + + ++ Gene presentdef Gene present but defective due to mutationPage 4 of 15(page number not for citation purposes)of all of the sequenced Pasteurellaceae for homologues ofall of these genes [29]. Table 1 shows that all of themon ancestor of H. influenzae and P. multocida, and thusof the Hin-subclade in Fig. 1, has been estimated to haveBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82lived about 270 million years ago (mya), and last com-mon ancestor of the entire family must be older still[30,31]. Thus the origin of the Pasteurellaceae is likely tohave long predated the origin of mammals (c.195 mya)and may be contemporaneous with the origin of tetrap-ods about 360 mya. If so, it is possible that these bacteriamoved into the respiratory tract and used the abundantDNA found there [32] almost as soon as the first respira-tory tracts evolved.What then explains the sporadic distribution of compe-tence in its descendants? Three of the five genomes from'non-transformable' species we analyzed carry obviousgenetic defects that would prevent DNA uptake. (Loss ofcomM in M. haemolytica would only prevent transforma-tion.) Each defect is unique and so must have arisen sincethe most recent divergence in its lineage. Furthermore, thesubstitution rate indicated by the scale bar on Fig. 1 allowsestimation of the minimum number of chain-terminatingand frameshift mutations expected to have accumulatedsince loss of a competence gene removed selection onother competence-specific genes. The scarcity of suchmutations in each of these strains (0, 1 or 2) suggests thatcompetence was lost quite recently. This is consistent alsowith the high densities and strong consensuses of the USSin all genomes except H. ducreyi. Frequent recent losses ofcompetence would also explain the reported variation incompetence within populations [3-6].Uptake signal sequences (USS) are not insertionsOne goal of this work was to use USS distribution to makeinferences about the evolution of DNA uptake specificity.However, the anomalously strong consensuses of H. influ-element rather than by point mutations in pre-existingsequences. Fortunately the mode of USS origin makes asimple prediction about the positions of gaps in sequencealignments. If individual H. influenzae USSs have arisen byinsertion, gaps should be seen when the segments con-taining these USS are aligned with homologous sequencesfrom genomes that diverged before USS arose. We usedthis prediction to test whether the many H. influenzaeUSSs in protein coding sequences arose in an ancestralPasteurellacean by insertion or by accumulation of pointmutations in the ancestral genes.Because of the evolutionary distance between H. influen-zae and species with no USS, the alignments were donebetween predicted amino acid sequences rather thannucleotide sequences. Segments of well-conserved H.influenzae proteins, centred on USS-encoded amino acids,were aligned with homologous protein segments fromEscherichia coli, Vibrio cholerae and Pseudomonas aeruginosa,whose genomes do not contain USS-like repeats. A typicalalignment is shown in Fig 2, along with a sketch of theevolutionary relationships of these bacteria.Of the 956 H. influenzae USS in protein-coding regions(65% of all USS), 158 were in protein segments that couldbe aligned with = 50% identity to homologues from allthree other species. Only 24% (115/474) of the individualE. coli, V. cholerae and P. aeruginosa proteins alignmentscontained gaps, and most gaps were outside of the seg-ment encoded by the USS core. Similar results were seenin alignments between homologues from outside thegamma-proteobacteria and the subset of H. influenzaeprotein sequences sufficiently well conserved to align wellExample gap analysis of a USS-homologous peptideFigure 2Example gap analysis of a USS-homologous peptide. A USS-encoded peptide and flanking region within the H. influen-zae folD gene (HI0609; amino acids 203–245), aligned with folD proteins from: E.coli (b0529; amino acids 203–246), Vibrio chol-erae (VC1942; amino acids 228–271), and Pseudomonas aeruginosa (PA1796; amino acids 203–246). The sketch at the right shows the phylogenetic relationships of these taxa [57].HI0609 DILVVAVGKPNLISGDWIKESAVVIDVGINRVD-GKLVGDVEFb0529 DLLIVAVGKPGFIPGDWIKEGAIVIDVGINRLENGKVVGDVVFVC1942 DILVVAVGKPNFIPGAWIKEGAVVVDVGINRLDTGKLVGDVEYPA1796 DLVVVAAGKPGLVKGEWIKEGAIVIDVGINRQADGRLVGDVEY*:::**.***.:: * ****.*:*:******   *::**** :AAGTGCGGTNo USSPage 5 of 15(page number not for citation purposes)enzae USSs (and other USSs) raised the concern that theymight have been produced by insertion of a replicating(results not shown). The homology between amino acidsthat are USS-encoded in H. influenzae and amino acids inBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82distant relatives confirms that USSs have arisen by nucle-otide substitutions in pre-existing sequences and not byinsertions of a replicative element.All Pasteurellacean genomes contain USS-like repeatsThe next step was to characterize the phylogenetic distri-bution of USS. Bakkali et al. found that the only overrep-resented short repeats in the P. multocida genome arevariants of the 9 bp H. influenzae USS core [13]. They alsofound the H. influenzae USS core to be highly overrepre-sented in the H. somnus and A. actinomycetemcomitansgenomes but did not survey other repeats. To avoid thebias of searching for a specific USS sequence, we extendedthis analysis by counting all 6–12 bp repeats in all eightgenomes (Table 2) and calculating the number of eachrepeat expected in a random-sequence genome of thesame size and base composition.Table 2 shows that all genomes had highly overrepre-sented repeats related to the H. influenzae USS. The mostcommon 9-mer repeats in the genomes of A. actinomyce-temcomitans, P. multocida, M. succiniciproducens and H.somnus are the H. influenzae USS core AAGTGCGGT andits reverse complement. All of the ten most abundant 8-mer, 9-mer and 10-mer repeats in these genomes also con-tain or closely overlap this 9-mer. We will refer to this asthe Hin-type USS. However the most frequent 9-merrepeats in the genomes of A. pleuropneumoniae and M.haemolytica differed from the Hin-type USS at the second,third and fourth positions (ACAAGCGGT rather thanAAGTGCGGT); we will refer to this as the Apl-type USS.The most abundant repeats in the H. ducreyi genome werenot recognizable USSs but simple palindromes andstrings of As and Ts, so Table 2 also gives the frequenciesnumbers were substantially lower than in the othergenomes. (Although the 10-mer AATAAGCGGT was themost common USS-like 10-mer repeat, ATAAGCGGT andTAAGCGGT were not among the 50 most frequent 9-mersand 8-mers.)Each genome was specifically checked for repeats of theother USS type. The frequencies of both types of 9 bp USSsper Mb sequence in all eight genomes are shown in Fig.3A. Only 4 copies per Mb would be expected in random-sequence genomes of the same base compositions.Although the minority USS type (e.g. Hin-type USS in A.pleuropneumoniae) is several-fold overrepresented in eachgenome, it is not significantly more frequent than other 9-mers sharing the global consensus ANNNGCGGT. Thuseach genome appears to have a predominant subclade-specific USS type.Fig. 3B shows, for each Pasteurellacean genome, the ratioof repeats perfectly matching each USS type to repeatswith single mismatches to that type. Genomes with Hin-type USSs resemble H. influenzae in having more perfectthan singly mismatched copies, despite the 27-fold greaternumber of possible sequences. The discrepancy is alsoseen for genomes with Apl-type USSs; with the exceptionof H. ducreyi, the ratio is substantially higher for the subc-lade-specific USS type than for the other type. The consist-ency of the pattern suggests that USS accumulation isshaped by similar forces in the different genomes.Detailed comparisons of USSsAs USSs are thought to function by binding to DNA recep-tors on the cell surface, bases at different positions in theUSS core would be expected to show consensus strengthsTable 2: Most common 8-, 9- and 10-mers in Pasteurellacean genome sequencesGenomea %G+C Size (Mb) Most common 10-merb (number, fold over-rep)Most common 9-merb (number, fold over-rep)Most common 8-merb (number, fold over-rep)Hin 38.1 1.8 AAAGTGCGGT (1115, 429X) AAGTGCGGT (1471, 175X) AAGTGCGG (1687, 63X)Aac 44.4 2.1 AAAGTGCGGT (1422, 384X) AAGTGCGGT (1760, 132X) AAGTGCGG (1863, 39X)Pmu 40.4 2.3 AAAGTGCGGT (700, 200X) AAGTGCGGT (927, 79X) AAGTGCGG (1013, 26X)Hso 37 2.1 AAAGTGCGGT (776, 273X) AAGTGCGGT (1216, 135X) AAGTGCGG (1446, 51X)Msu 42.5 2.3 AAAGTGCGGT (1297, 333X) AAGTGCGGT (1485, 111X) AAGTGCGG (1622, 46X)Apl 41.4 2.2 ACAAGCGGTC (429, 187X) ACAAGCGGT (742, 68X) CAAGCGGT (1361, 36X)Mha 41 2.7 ACAAGCGGTC (506, 181X) ACAAGCGGT (973, 70X) CAAGCGGT (1636, 48X)Hduc 38.2 1.76 TTTTGCAAAA (106, 9.6X)AATAAGCGGTc (95, 21X)AACAAGCGGTc (85, 31X)AATAAAAAA (251, 2.8X)ACAAGCGGTc (199, 23X)AAAAATAA (680, 2.3X)CAAGCGGTc (464, 16X)a Hin: H. influenzae; Aac: A.s actinomycetemcomitans; Pmu: P. multocida; Hso: H. somnus; Msu: M. succiniciproducens; Apl: A. pleuropneumoniae; Mha: M. haemolytica; Hdu: H. ducreyi.b The first number in parentheses is the combined number of copies of the sequence and its reverse complement. The second number in parentheses is the fold over-representation of each repeat compared to a random-sequence genome of the same size and base composition.c The additional entries for H. ducreyi are the copy numbers of the most frequent USS-like repeats.Page 6 of 15(page number not for citation purposes)of the most common USS-like 8, 9 and 10-mers for thisgenome. These resembled the Apl-type USS but their copyreflecting their differing contributions to this DNA-pro-tein binding. Sequence logos were used to visualize theBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82representation of each base at each position of the USS(Figs. 4 and 5) [33]. In these logos the relative heights ofthe A, G, C and T in each stack shows the frequencies ofthe bases at that position, and the overall height of eachstack of letters reflects the strength of the consensus at thatposition (the information content). The height of thestack is especially sensitive to minor changes in the fre-quency of a very frequent base (e.g. if the frequency of themost common base falls from 1.0 to 0.9 the height fallsfrom 2.0 to 1.6).Fig. 4A shows the differences in consensus strengths at theUSS core positions. In all genomes with Hin-type USSsmost core positions appear to have roughly equal consen-sus strengths, suggesting that all make similar contribu-tions to binding specificity. The exceptions are the final T,which has a weaker consensus in H. influenzae and H. som-nus, and the first G in M. succiniproducens. For genomeswith the Apl-type USS the first two positions of USS coresshow a weaker consensus than the other positions, sug-gesting that they may make a lesser contribution to thespecificity of DNA binding and uptake. Confirmation ofthese predictions must await identification and character-ization of the proteins or other molecules that interactwith the USS at the cell surface.The H. influenzae and A. actinomycetemcomitans USSs havebeen shown to also share conserved motifs (segments 2and 3) on the 3' side of the USS core [9,12]. The impor-tance of segment 2 was experimentally demonstrated byDanner and coworkers, who showed that USS-containingDNA fragments ethylated at bases in this region were nottaken up by competent H. influenzae cells [15]. The func-tions of these positions in DNA uptake are not known;they may be additional sites of contact with the DNAreceptor, or they may be involved in DNA bending orkinking during uptake.Fig. 4B shows the consensuses of positions flanking thecore USSs in each of the eight genomes. As viewed in thestandard orientation, all USS consensuses have an AT-richmotif just 3' of the core (segment 2; positions 22–27 asnumbered in Fig. 4B), with the first bases usually 'A's andthe final 3 or 4 bases 'T's. A second AT-rich motif is seenfurther downstream (segment 3). In the Hin-type USS this6nt-motif segment consists primarily of Ts, and is centred12 positions to the right of segment 2. In A. pleuropneumo-niae and M. haemolytica, segment 3 extends slightly fartherto the left and substantially farther to the right, and hasthe more complex consensus AAAATTTTGCAAAT.Although the H. ducreyi USS consensus in segments 2 and3 resembles the Apl-type motifs, it is much weaker.Together with the lower frequency of USS in its genome,competence genes, this suggests a relatively ancient loss ofability to take up DNA.The consensuses in segments 2 and 3 of the A. pleuropneu-moniae and M. haemolytica USSs were particularly strongand extensive. To compare their strengths to that of thecore USS we repeated the above analysis in reverse. Wechose the nine bases making the strongest contribution tosegment 2 and segment 3 (ATTTNNNNNNNNNTTTGC)or to segment 3 alone (TTTTGCAAA) and identified andaligned all A. pleuropneumoniae genomic segments con-taining them (560 and 454 segments respectively). Theresulting logos (Fig. 5A and 5B) show that many of thesegments bearing these motifs also contained all but thefirst two bases of the Apl-type USS core. The weak correla-tion of the first two positions of the core with the flankingsegments may mean that these positions play a lesser rolein USS function than the rest of the core, with its largersegment 3 making a greater contribution to the bindingspecificity. A logo using only the 164 sequences with 12matches to segments 2 and 3 was even more effective,recovering the full core consensus (Fig. 5C). Takentogether, these analyses suggest that, at least in A. pleurop-neumoniae, the motifs in segments 2 and 3 may be asimportant for DNA uptake as the USS core.USS in H. parasuisA recent paper reported that H. parasuis has the core USSGAGTTCGGT, which differs from both the Hin and Apltypes[26]. However this conclusion was based on analysisof a single putative USS in a cloned 413 bp fragment. Wehave examined all the available H. parasuis sequences(86,701nt, mainly in ORFs) and find, in addition to theone copy of this repeat described by Bigas et al., four cop-ies of the Hin-type USS, fourteen copies of the Apl-typeUSS, and seventeen copies of sequences differing at singlepositions from the Apl-type USS. This suggests that H. par-asuis has an Apl-type USS, which would be consistent bothwith previous phylogenetic analysis placing it in astrongly supported subclade with M. haemolytica and A.pleuropneumoniae [17,34] and with the ability of A. pleu-ropneumoniae DNA to efficiently transform H. parasuis[35].H. influenzae and A. pleuropneumoniae recognize subclade-specific USSsA. actinomycetemcomitans (Hin-type USS) has already beenshown to preferentially take up its own and H. influenzaeDNAs [12], but for most of the other species the role of theputative USS in DNA uptake could not be directly testedbecause no competent isolate has been identified. How-ever A. pleuropneumoniae strain HS143 (serotype 15) hasrecently been shown to be much more competent thanPage 7 of 15(page number not for citation purposes)and the presence of inactivating mutations in three of its other strains (J. Bossé, manuscript in preparation), allow-ing us to test its uptake specificity by three different exper-BMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82iments. Each confirmed that competent A.pleuropneumoniae cells preferentially take up DNA frag-ments containing the Apl-type USS.The solid bars in Figure 6A and 6B show measurements ofuptake by competent H. influenzae and A. pleuropneumo-niae cells of radiolabelled 220 bp DNA fragments contain-ing synthetic H. influenzae and A. pleuropneumoniae USSs.These USSs were designed to contain the most commonbase at each position of the extended USSs describedabove; a control fragment contained a randomized ver-sion of the H. influenzae USS sequence. As expected, H.influenzae took up about 1500-fold more DNA containingits USS than control DNA (Fig. 6A; note the log scale). Thefunction of the Apl-type putative USS was confirmed; A.pleuropneumoniae took up about 17-fold more DNA withits USS than control DNA (Fig. 6B). Each species also tookup substantially less DNA containing the heterologousUSS type than its own type (only about twice as much ascontrol DNA), confirming that the DNA uptake machin-ery discriminates between the two types.in Fig. 6A and 6B show uptake of radiolabelled chromo-somal DNAs by competent H. influenzae and A. pleurop-neumoniae cells. In this assay H. influenzae took up 50-foldmore H. influenzae DNA than the control E. coli DNA (Fig.6A), and A. pleuropneumoniae took up about 37-fold moreA. pleuropneumoniae DNA than E. coli DNA (Fig. 6B). Inboth chromosomal and synthetic-USS uptake experi-ments A. pleuropneumoniae took up substantially less DNAthan did H. influenzae, consistent with its lower transfor-mation frequency.Consideration of the relative densities of the two USStypes in the three genomes (H. influenzae: A. pleuropneu-moniae: E. coli; 198:8:1 (Hin-type USSs) and 4.4:48:1 (Apl-type USSs)) allows clarification of the degree to whicheach species takes up DNA of the other type. First, the highdensity of Hin-type USS in the H. influenzae genomemeans that most chromosomal DNA fragments (≥ 50 kblong) would have contained at least several USS, reducingthe contribution of each USS to uptake. Second, the pres-ence of heterologous USS in the two Pasteurellaceangenomes can explain H. influenzae's 6-fold higher uptakeUSS frequencies in sequenced Pasteurellacean genomesFigur  3USS frequencies in sequenced Pasteurellacean genomes. Red: Hin-type USSs (AAGTGCGGT); blue: Apl-type USSs (ACAAGCGGT). A. Frequencies of 9 bp core USSs of each type per Mb of genome. B. Ratios of perfect to singly mismatched 9 bp USS cores.0200400600800Hin Aac Pmu Hso Msu Apl Mha HduGenomeUSS per MbA.012GenomePerfect:one-off ratioB.Hin Aac Pmu Hso Msu Apl Mha HduPage 8 of 15(page number not for citation purposes)Uptake of chromosomal DNA may provide a more bio-logically relevant measure of specificity. The dashed barsof A. pleuropneumoniae DNA than E. coli DNA, but is likelyinsufficient to explain A. pleuropneumoniae's 32-foldBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82higher uptake of H. influenzae DNA than E. coli DNA.These results suggest that the A. pleuropneumoniae uptakemachinery does indeed weakly recognize the Hin-typeUSS, and do not preclude a similar overlap in specificityby the H. influenzae uptake machinery.Figure 7 shows the extent to which cells preferentially takeup genetically marked conspecific DNA in the presence ofcompeting DNA from their own strain or another species.This is a more sensitive measure of uptake bias than theuptake of pure DNAs tested above. Fig. 7A shows theresults of uptake-competition assays using H. influenzaecells and a constant amount of H. influenzae chromo-somal DNA carrying a novobiocin resistance allele. Asexpected, unmarked H. influenzae DNA competedstrongly but B. subtilis DNA, which does not contain over-represented USS-like repeats, did not [36]. A. pleuropneu-ence to both H. influenzae DNA and B. subtilis DNA. Theseresults confirm that the DNA uptake machineries of bothH. influenzae and A. pleuropneumoniae discriminatestrongly in favour of DNAs containing their own USStype. H. parasuis DNA was also tested; it did not competefor uptake by H. influenzae (Fig. 7A), but competed withA. pleuropneumoniae DNA for uptake by A. pleuropneumo-niae to an extent consistent with the density of Apl-typeUSS in its DNA.We did not test whether cells could discriminate betweenDNAs from species in the same subclade. However, as anearlier measure of relatedness among the Pasteurellaceae,Albritton et al. examined the ability of DNAs from variousspecies to compete with H. influenzae DNA for uptake bycompetent H. influenzae cells [37]. The ability to competefor uptake correctly predicted the USS distributions weWebLogos for USSs and surrounding sequence in 8 genomesFigure 4WebLogos for USSs and surrounding sequence in 8 genomes. A. Logos based on 9 bp segments with perfect or one-off matches to the 9 bp USS. B. Logos based on 50 bp segments with perfect matches to the 9 bp USS.HinAacAplHsoHduMhaMsuPmuA. B.Segment 2 Segment 3Page 9 of 15(page number not for citation purposes)moniae DNA did not compete for uptake. Fig. 7B showsthat A. pleuropneumoniae took up its own DNA in prefer-have found: DNAs from A. actinomycetemcomitans and P.multocida (Hin subclade) competed strongly (54% andBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/8244% as well as H. influenzae DNA), but DNA from A. pleu-ropneumoniae (Apl subclade) competed only poorly (7%).Although the other sequenced species were not tested, thecompetition shown by DNA of non-sequenced Pasteurel-lacean species is likely to predict the USS types they con-tain. Thus the strong competition Albritton et al. observedby H. parainfluenzae, H. aphrophilus, H. paraphrophilus andP. pneumotropica DNAs suggests that they likely carry Hin-type USSs. Consistent with this, H. parainfluenzae isknown to have Hin-type USSs [15], and Christensen et al.'srRNA and protein trees place P. pneumotropica close to P.multocida with reasonable bootstrap support; their rRNAtree also places H. aphrophilus and H. paraphrophilus closeto H. influenzae with modest bootstrap support. In con-trast, Albritton et al. found that DNAs of P. ureae, A. lig-nieresii and A. equuli competed very poorly with H.influenzae DNA. These taxa are closely linked to A. pleurop-neumoniae in Christensen et al.'s rRNA tree, and A. lignier-esi is the sister taxon to A. pleuropneumoniae in theirprotein trees, supporting the hypothesis that they haveApl-type USSs [16,17].The shared features of the Pasteurellacean USS types maydomain of the as-yet-unidentified DNA receptor protein,and are similar in length to the 10 bp Neisseria core. Theconservation of segment 2 and segment 3 in the Pasteurel-laceae is intriguing, as conserved flanking motifs are notseen in Neisseria. It may be significant that the spacingsbetween the USS core, segment 2 and segment 3 corre-spond roughly to single turns of helical DNA. We know itcannot be the case that H. influenzae cells initiate uptakeby threading a DNA end through a membrane pore,because they efficiently take up covalently closed plas-mids [38]. However DNA molecules are too highlycharged and too stiff (persistence length about 50 nm or150 bp) to simply pass sideways through the outer mem-brane. Together the USS core plus flanking motifs mayallow the DNA to be sharply kinked (perhaps by strandseparation), presenting a compact cross-section for mem-brane transit. Detailed understanding of the function ofUSSs will require more complete experimental studies ofbinding and incorporation of target DNA sequences.ConclusionThe eight Pasteurellacean species we analyzed fall intotwo robust subclades. The genomes of all these bacteriaWebLogos for 50 bp segments of the A. pleuropneumoniae genomeFigure 5WebLogos for 50 bp segments of the A. pleuropneumoniae genome. The highlighting indicates the positions used to choose sequences for analysis.A. Logos for segments containing the motif TTTTGCAAA. B. Logos for segments containing the motif ATTTNNNNNNNNNTTTGC. C. Logos for segments containing the motif AATTTNNNNNNNNNTTTGCAA.A.Segment 2 Segment 3B.C.Page 10 of 15(page number not for citation purposes)reflect generalized features of the DNA uptake process.The 9 bp USS cores may match the size of the recognitioncontain homologues of all the H. influenzae genes knownto be needed for DNA uptake, some of which haveBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82Page 11 of 15(page number not for citation purposes)Uptake of synthetic USSs or chromosomal DNAsFigur 6Uptake of synthetic USSs or chromosomal DNAs. The solid bars show uptake of 220 bp PCR fragments containing syn-thetic USS with the consensus sequences of H. influenzae (Hin), A. pleuropneumoniae (Apl) or randomized H. influenzae USS types. The dashed bars show uptake of chromosomal DNAs from H. influenzae (Hin), A. pleuropneumoniae (Apl) or E. coli. Error bars show the standard deviations of 3 replicate experiments, except for A. pleuropnemoniae in A, which is from 4 replicate experiments. A. Uptake by H. influenzae. B. Uptake by A. pleuropneumoniae.USS or DNA typeUptake of chromosomal DNA(ng/109cfu)USS or DNA  typeUptake of synthetic USS(ng/109 cfu)0.0020.020.22.020Hin Apl Random/E. coliHin Apl Random/E. coliB.0.010.11101000.010.1110100Uptake of synthetic USS(ng/109cfu)Uptake of chromosomal DNA(ng/109cfu)A.0.0020.020.22.020BMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82recently been inactivated by mutation. All of thesegenomes also contain high densities of genetically stablerepeats, either the well-characterized H. influenzae USS ora related sequence, in each case comprising a 9 bp coreand two adjacent AT-rich segments. The distribution ofthe Hin-type and Apl-type USSs corresponds to the twoPasteurellacean subclades. Competent members of thesesubclades discriminate between the two USS types, eachpreferring to take up DNA containing the USS typical of itsown genome.Taken together, these findings are consistent with the fol-lowing model of the evolution of competence in the Pas-teurellaceae: The ancestor of the sequenced Pasteurellaceaepossessed a complete set of functional competence genesand was naturally competent, taking up DNA by a mech-anism very similar to that used by H. influenzae today. Theancestral genome contained many USSs; these may orthe USS core and included the AT-rich segment 2 andmuch of segment 3. During the initial diversification ofthe Pasteurellacean subclades the uptake specificity andUSS consensus changed in parallel in one or both line-ages. This divergence of genomic USSs may have beeneffectively complete before the divergence of thesequenced species within each subclade, with USS specif-icities remaining stable since then, although the existenceof Pasteurellaceae with other diverged uptake specificitiescannot be ruled out. Because the USS consensuses withineach subclade are so similar, USS specificity will not ena-ble competent bacteria to distinguish between DNAsderived from different species within their subclade.Because many of these DNAs are otherwise sufficientlydiverged that recombination is not only inefficient buttoxic [37,39], forces other than exclusion of non-self DNAmay be responsible for uptake specificity.What are the implications for other bacterial families? Wesuggest that the evolutionary history of competence oftenfollows the pattern shown in Fig. 8. In this model, theancestors of many bacterial families were naturally com-petent but competence has been and continues to be fre-quently lost. Mutations causing loss of competence havenot always been strongly selected against, and sometimesmay have been actively favoured, so non-competent line-ages often persist. However, over the long term the non-competent lineages are selected against, so that all extantbacteria have recent ancestors who were competent. Thishypothesis is consistent not only with our family-levelanalysis but with the extensive evidence of sporadic distri-bution of competence within individual species [3-6]. Thepattern is similar to that seen for the mismatch repair sys-tem, where mutants with defects in mutation preventioncan experience a short-term advantage but are eventuallyeliminated by selection against accumulating deleteriousmutations [40].Many questions remain unanswered. How deep is theancestry of competence? Are some bacterial familiesancestrally not competent? Have some modern speciescompletely lost competence? Do genes introduced by con-jugation or transduction ever restore competence to non-competent lineages? Thanks to the ever-increasing availa-bility of new genome sequences, answers to these ques-tions will soon be within reach.MethodsGenome and gene sequencesChoice of sequencesComplete and annotated genome sequences were availa-ble through NCBI for a single isolate each of P. multocida,M. succiniciproducens and H. ducreyi [30,22,23]. SequencesDouble-reciprocal plots of uptake competition assaysFig r  7Double-reciprocal plots of uptake competition assays. Double-reciprocal plots of uptake competition assays. Cx/Co: ratio of competing DNA to genetically marked self DNA. To/Tx: ratio of number of transformants in the presence and absence of competing DNA Competing DNAs: blue diamonds, H. influenzae; red squares, A. pleurop-neumoniae; black triangles, B. subtilis; green circles, H. parasuis. A. Competition in H. influenzae. B. Competition in A. pleu-ropneumoniae.H. influenzae competitions0510To/TxHinAplBsuHpaA. pleuropneumoniae competitions05100 2 4 6 8 10Cx/CoTo/TxPage 12 of 15(page number not for citation purposes)may not have been simpler than the USSs in its descend-ants, but likely had the common motif ANNNGCGGT inof four H. influenzae isolates were available [41]; we usedthe fully assembled and annotated sequence of the RdBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82strain [42]. Sequencing is in progress for two H. somnusisolates, we used the more complete 129-PT sequence.Four incomplete A. pleuropneumoniae genome sequenceswere listed at NCBI; however we used our nearly completesequence of strain L20 (serotype 5 b, sequence availablefrom JN on request). The genome sequences of M. haemo-lytica and A. actinomycetemcomitans are also in progressand not yet available at NCBI.Complete genome sequences with annotations wereretrieved from the NCBI website [43] for H. influenzae RdKW20 (NC_000907.gbk), P. multocida subsp. multocidastr. Pm70 (NC_002663.gbk), M. succiniciproducensMBEL55E (NC_006300.gbk), H. ducreyi 35000 HP(NC_002940.gbk), E. coli K12 (NC_000913.gbk), V. chol-erae N16961 (NC_002505.gbk and NC002506.gbk) andP. aeruginosa PA01(NC_002516.gbk). Unfinishedgenome sequences were obtained for A. actinomycetem-comitans from [44] and for H. somnus 129-PT from [45].Preliminary sequence data for M. haemolytica wereobtained from the Baylor College of Medicine HumanGenome Sequencing Center [46,47]. Open reading framesfor genome sequences lacking annotation were identifiedfrom the draft sequence using the GLIMMER softwarepackage (now available at [48]).Phylogenetic analysisPhylogenetic analysis used the amino acid sequences ofthe following 12 H. influenzae genes and their best homo-logues in the other 7 Pasteurellaceaen genomes and in E.coli: gapdH (HI0001), lepA (HI0016), ffh (HI0106), serS(HI0110), secD (HI0240), dapA (HI0255), ruvB(HI0312), xerC (HI0676),ispB (HI0881), secA (HI0909),crp (HI0957), and dnaJ (HI1238). Homologues weresequences from the 9 genomes were aligned using CLUS-TALW with output in PHYLIP format [50]. The alignedsequences were inspected, the ends were trimmed toremove sequence missing in any of the 9 genomes, andthe alignments of the 12 genes were concatenated to pro-duce a single long alignment.Phylogenetic analysis of the concatenated alignment usedthe PHYLIP software package [51]. ProML analysis usingmaximum likelihood with the JTT method and a gamma-plus-invariant-sites distribution of rates across sitesyielded a predicted tree with estimated phylogenetic dis-tances. SeqBoot was then used to produce 100 datasets bybootstrapping resampling; these were put into ProML togenerate phylogenetic trees. The final bootstrap analysiswas done using the program Consense and the bootstrapvalues were added to the tree generated with the completesequence above.Homology of USS-encoded peptidesBLAST searches were used to identify those USS-contain-ing H. influenzae genes that had homologues in all of E.coli, V. cholerae and P. aeruginosa. ClustalW was used toalign the homologous protein sequences, with the defaultpenalties of 10 for gap opening and 0.2 for gap extension.Analysis was restricted to 43aa segments centred onamino acids encoded by the USS core that showed >50%amino acid identity across all homologues. All gapswithin these alignments were tabulated.Repeat analysisThe Perl program repeat_finder was developed to searchgenome sequences for abundant short DNA sequences(code available at [52]). It was used to tabulate the occur-rences of the 20 most abundant 6-, 7-, 8-, 9-, 10-, 11-, and12-mers for each of the 8 Pasteurellacaean genomes,along with the number of each expected for a random-sequence genome of that size and nucleotide composi-tion.All occurrences of the 9 bp putative USS core for each spe-cies were identified, and 50-bp sequence segments con-taining the core plus 11 bases upstream and 30 basesdownstream were aligned. The program WebLogo [7,53])was used to visualize the consensus for each USS. Similaranalyses were done for each genome using all singly mis-matched occurrences of the 9 bp core, and for A. pleurop-neumoniae using consensus sequences derived from thetwo flanking regions.Bacterial strains and culture conditionsA. pleuropneumoniae serotype 15 (strain HS143) and H.influenzae Rd (strain KW20) were grown in Brain HeartModel for the evolution of competenceFigure 8Model for the evolution of competence.competent strainnon-competent strainSpecies 1Species2Page 13 of 15(page number not for citation purposes)identified using the BLASTP program (E-val < 10-50) in theBLAST package [49]. For each gene, the amino acidInfusion broth (Difco) supplemented with the recom-mended concentrations of NAD and hemin (H. influenzaeBMC Evolutionary Biology 2006, 6:82 http://www.biomedcentral.com/1471-2148/6/82only), and were made competent by transfer of exponen-tially growing cells to MIV starvation medium asdescribed for H. influenzae[54]. Aliquots of competentcells were stored at -80°C and thawed immediately beforeuse.DNA labelingChromosomal DNAs of H. influenzae Rd and A. pleurop-neumoniae were labeled by nick-translation with alpha-33P-dATP to specific activities of 2 × 107 cpm/μg. Frag-ments of about 200 bp centered synthetic USSs (USS-Hin:CCCAAAGTGCGGTTAATTTTTTACAGTATTTTTGGGTTC-GAAAT; USS-Apl: GGAAACAAGCGGTCAAATTT-GCCGAAAATTTTGCAAATTGGTACCT; USS-Ran:TCTTGTTAGAATCTGAGTGTTATTTAAAT) were PCR-amplified from clones in pGEM [55] using primers withBglII ends. The amplified fragments were cut with BglIIand end-labeled with Klenow polymerase using alpha-33P-dATP, to specific activities of 106–107 cpm/μg.DNA uptakeCompetent cells of H. influenzae strain KW20 and A. pleu-ropneumoniae strain HS143 (1.0 ml; ~1 × 109 cfu) wereincubated with 150 ng of labeled chromosomal DNA or20 ng of labeled PCR fragment for 15 minutes at 37°C,followed by 5 minutes incubation with DNase I at 1 μg/ml. Cells were then washed three times at room tempera-ture by pelleting and resuspension in 1.0 ml of MIV, andthe radioactivity of the pellets was counted.Transformation-competition experimentsCompetent cells of H. influenzae strain KW20 and A. pleu-ropneumoniae strain HS143 (0.2 ml) were incubated for 15minutes at 37°C with 100 ng of genetically marked con-specific DNA (MAP7 DNA for H. influenzae [54] andsodC::Kan DNA for A. pleuropneumoniae [25]) mixed with100, 300, or 900 ng of competing DNA (H. influenzaeKW20, A. pleuropneumoniae HS143, B. subtilis or H. par-asuis (strain Nagasaki) DNA). DNaseI was then added at1.0 μg/ml for a further five minutes and cells were thendiluted and plated on supplemented BHI plates contain-ing 2.5 μg.ml novobiocin (H. influenzae) or 25 μg/ml kan-amycin (A. pleuropneumoniae). Data were plotted using thedouble-reciprocal method of Sisco and Smith [56].Authors' contributionsRJR, WAF, JB and JHEN were involved in the conceptionand design of the study. RJR carried out the DNA-uptakeand competition experiments, drafted the manuscript andproduced the figures. WAF carried out most of the bioin-formatics analyses. JB analyzed the H. parasuis sequences.ADSC identified and analysed the competence genes ineach species. JHEN participated in the bioinformaticsthe manuscript, and all read and approved the final man-uscript.AcknowledgementsFunding for this work has been provided to J.H.E.N. and W.A.F. through the National Research Council of Canada Genomics and Health Initiative, to R.J.R. through the Canadian Institutes for Health Research and National Institutes of Health Grant RO1 GM60715, and to JSK/JB from NRCBC joint S&T. We thank Patrick Keeling for advice on phylogenetic methods, Sarah Highlander for alerting us to the USS-like sequences in M. haemolytica, Jan MacInnis for H. parasuis DNA, and Rob Beiko and Barry Hall for helpful dis-cussions. Assembly and preliminary annotation of the A. pleuropneumoniae L20 genomic sequence was done by Simon Foote. The H. somnus genome sequence data were produced by the US Department of Energy Joint Genome Institute [45]. Preliminary M. haemolytica sequence data was obtained from the Baylor College of Medicine Human Genome Sequencing Center website [46], supported by USDA/NRICGP grant 00-35204-9229 to Sarah Highlander and George Weinstock at the BCM-HGSC. We thank the Actinobacillus Genome Sequencing Project, and Bruce A. Roe, Fares Z. Najar, Allison Gillaspy, Sandra Clifton, Tom Ducey, Lisa Lewis and D.W. Dyer for the A. actinomycetemcomitans sequence data; this project is sup-ported by a USPHS/NIH grant from the National Institute of Dental Research.References1. Chen I, Dubnau D: DNA uptake during bacterial transforma-tion.  Nat Rev Microbiol 2004, 2(3):241-249.2. Solomon JM, Grossman AD: Who's competent and when: regu-lation of natural genetic competence in bacteria.  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