Evidence for Vertical Inheritance and Loss of the Leukotoxin Operon in Genus Mannheimia

Evidence for Vertical Inheritance and Loss of the Leukotoxin Operon in Genus

Mannheimia

Jesper Larsen,1 Anders G. Pedersen,2 Henrik Christensen,1 Magne Bisgaard,1 Øystein Angen,3 Peter Ahrens,3

John E. Olsen1

1 Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederiksberg C,

Denmark2 Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Lyngby, Denmark3 National Veterinary Institute, Technical University of Denmark, Bulowsvej 27, DK-1790 Copenhagen V, Denmark

Received: 19 March 2006 / Accepted: 30 January 2007 [Reviewing Editor: Dr. David Guttman]

Abstract. The Mannheimia subclades belong to thesame bacterial genus but have taken divergent pathstoward their distinct lifestyles. M. haemolytica +M. glucosida are potential pathogens of the respiratorytract in the mammalian suborder Ruminantia,whereas M. ruminalis, the supposed sister group, livesas a commensal in the ovine rumen.We have tested thehypothesis that horizontal gene transfer of the leuko-toxin operon has catalyzed pathogenic adaptation andspeciation ofM. haemolytica+M. glucosida, or othermajor subclades, by using a strategy that combinescompositional and phylogenetic methods. We showthat it has been vertically inherited from the lastcommon ancestor of the diverging Mannheimia subc-lades, although several strains belonging to M. rumi-nalis have lost the operon. Our analyses support thatdivergence withinM. ruminalis following colonizationof the ovine rumen was very rapid and that functionaldecay ofmost of the leukotoxin operons occurred earlywhen the adaptation to the rumen was fastest, sug-gesting that antagonistic pleiotropy was the maincontributor to losses in the radiating lineages of M.ruminalis. To sum up, the scenario derived from theseanalyses reflects two aspects. On one hand, it opposesthe hypothesis of horizontal gene transfer as a catalystof pathogenic adaptation and speciation. On the otherhand, it indicates that losses of the leukotoxin operons

in the radiating lineages ofM. ruminalis have catalyzedtheir adaptation to a commensal environment andreproductive isolation (speciation).

Key words: Mannheimia — Leukotoxin — Verticalinheritance — Gene loss

Introduction

The leukotoxin (LktA) protein of Mannheimiahaemolytica belongs to the E. coli HlyA-like sub-family of cytotoxic RTX (repeats in toxin) proteinspresent in a range of Gram-negative bacteria (Loet al. 1987). These RTX proteins all undergo post-translational fatty acylation and conformationalchanges as part of maturing their biological behavior(Welch 2001). The subfamily is characterized by aconserved RTX domain (GGXGXDX(L/F/I)X; sin-gle-letter amino acid code) that is shared by mostsubstrates of the type I secretion system and by apropensity for multiple interactions with receptorswhich define their functional properties (Welch 2001).The leukotoxin (lkt) operon codes for four proteins:an internal acyltransferase, encoded by lktC (Lo et al.1987); a structural RTX toxin, encoded by lktA (Loet al. 1987); an inner membrane protein with acytoplasmic ATP-binding cassette (ABC) domain,encoded by lktB, which pumps out LktA protein viainteraction with the C terminus of LktA (Highlander

Jesper Larsen and Anders G. Pedersen contributed equally to this

work.

Correspondence to: Jesper Larsen; email: [email protected]

J Mol Evol (2007) 64:423–437DOI: 10.1007/s00239-006-0065-3

et al. 1989); and a membrane fusion protein, encodedby lktD, which forms a bridge between the inner andouter membranes (Highlander et al. 1989). Thegenes for these four proteins are physically adjacenton the chromosome and are transcribed as lktCAor lktCABD messages (Strathdee and Lo 1989;Highlander et al. 1990).

LktA is essential in both evasion and exploitationof the adaptive immune system during pulmonaryinfection (Petras et al. 1995; Tatum et al. 1998;Highlander et al. 2000). The LktA protein elicits anumber of responses in ruminant target cells derivedfrom the pluripotent hematopoietic stem cell,including polymorphnuclear leukocytes (PMNs;neutrophils), lymphocytes, macrophages, and redblood cells (Kaehler et al. 1980; Shewen and Wilkie1982; Chang et al. 1986). The biological effectsagainst leukocytes result from activation and apop-tosis at sublytic doses or membrane disruption andleakage of content at lytic doses (Jeyaseelan et al.2001; Deshpande et al. 2002; Leite et al. 2002).

Incorporation of foreign DNA is a major force inthe evolution of gene content of bacterial species(Koonin et al. 2001; Boucher et al. 2003). Theseevents are generally thought to catalyze adaptiveevolution by one of two molecular mechanisms: (i)RecA-dependent homologous recombination be-tween large repeats (‡25 bp) that may be very farapart in the chromosome; or (ii) illegitimate recom-bination between closely spaced repeats. Underhomologous recombination, gene transfer events canincrease the frequency of beneficial alleles via selec-tive sweeps leading to increased fitness of therecipient within its current niche (Lawrence 2002).This mechanism is constrained by the evolutionarydistance (DNA sequence divergence) between the twomating partners and the cellular machineries involved(Vulic et al. 1997, 1999). Independent of homologousrecombination, evolutionary novelty can be accom-plished by incorporation of nonhomologous genes oroperons via illegitimate recombination in a processknown as horizontal gene transfer (HGT), allowingthe recipient to exploit niches that are controlled byunpredictable selective processes (Lawrence and Roth1996; Lawrence 1997, 1999). In the most extremecases of HGT, the recipient and its maternal parentbecome reproductively isolated, supporting a role forHGT as a catalyst of speciation (Lawrence 2002).

A number of observations suggests that the lktoperon from genus Mannheimia has been acquired byHGT leading to pathogenic adaptation and specia-tion. An early study on the distribution of the lktoperon was made by Burrows et al. (1993), whofound that the operon was present in the speciesM. haemolytica and M. glucosida and in the moredistantly related taxon [Pasteurella] trehalosi. TheM. haemolytica + M. glucosida group forms one of

the most recently diverged subclades within genusMannheimia based on 16S rRNA sequences, and itsspecies are all potential pathogens in the mammaliansuborder Ruminantia (Angen et al. 1999). Interest-ingly, the CD11a/CD18 (aLb2) subunits of the lym-phocyte function-associated antigen-1 (LFA-1) wereidentified as a Ruminantia-specific receptor for LktAon leukocytes (Lally et al. 1997; Ambagala et al.1999; Li et al. 1999; Jeyaseelan et al. 2000), sup-porting that HGT of the lkt operon could have actedas a mechanism of pathogenic adaptation and speci-ation. The evolution of the lkt operon was revisitedby Davies et al. (2002), who used phylogeneticmethods to compare 6952 bp of the lkt operon fromM. haemolytica, M. glucosida biogroup 3B, and [P.]trehalosi and noted that the ancestral bovine operonfrom M. haemolytica has been acquired by HGTfrom a more distantly related donor. The reality ofthis HGT event should be questioned, however, asthe data said to support Mannheimia�s gain of the lktoperon are ambiguous. For example, the factthat M. ruminalis, the supposed sister group ofM. haemolytica + M. glucosida, lives as a commensalin the ovine rumen (Angen et al. 1999) supportsa HGT event into the last common ancestor ofM. haemolytica + M. glucosida (Fig. 1), but thepattern could also be due to altered selection on genesthat are involved in evasion and exploitation of theimmune system and consequent loss of the lkt operonin the M. ruminalis subclade.

In this article, we rigorously test the hypothesisthat the ancestor of M. haemolytica + M. glucosidagained the lkt operon via HGT by using a strategythat combines compositional and phylogeneticmethods. First, we identify bifurcation order of theMannheimia subclades, allowing the lktA genotypeand the corresponding b-hemolytic phenotype to bemapped and compared with transitions in key traits(e.g. pathogenicity and niche utilization). Then wetest for the origin of the lkt operons by inferring therelationships of partial lktA sequences with homolo-gous sequences from other Gram-negative bacteria.Finally, we rank genes according to their convergenceto the average genome signature of M. haemolyticabased on the relative 3:1 dinucleotide bias.

Materials and Methods

Taxa Used

For this study we used 58 strains to represent the diversity within

genusMannheimia based on phenotypic characters (biogroups) and

geographic origin (see Table 1 for a description of strains used in

this study, along with their accession numbers). However, it is

possible that the samples are biased because certain geographic

areas and certain organismal groups have been more extensively

studied than others and therefore are overrepresented in out data

424

sets (e.g., strains belonging to M. ruminalis have been isolated

exclusively from the United Kingdom). Most taxa in our analyses

have been represented in previous studies by Angen et al. (1997a–c,

1999) and Blackall et al. (2001), and ribotypes and electrophoretic

types are given in those publications.

Analysis of Genes, Pseudogenes, and Remnant DNASequences

We analyzed the distribution of the lkt operons in genus Mann-

heimia using Southern blot. We constructed a probe in the +845/

+1302 region of lktA fromM. haemolytica strain PHL213 by using

the forward primer manpop_UP (5¢-CCAAAGCCGTTTCTT

CTTACA-3¢) in conjunction with the reverse primer man-

pop_DOWN (5¢-TAACGGGCRTCGTAACCATT-3¢), which

were designed from published sequences. The reaction conditions

were 2.5 U Taq polymerase, 16 mM (NH4)2SO4, 67 mM Tris–HCl,

0.01% Tween-20, 2.5 mM Mg2SO4, each primer at 0.5 mM, and

each nucleotide at 0.1 mM. The cycling conditions were initial

denaturation at 94�C followed by 25 cycles at 94�C for 30 s, 52�Cfor 30 s, and 72�C for 30 s, finishing with extension at 72�C for 10

min. The PCR product was labeled with digoxigenin-11-dUTP

using the Random Primed DNA Labeling Kit according to the

manufacturer�s instructions (Roche).

To determine inactivation of the lkt operons, we screened the

strains for the b-hemolytic phenotype on sheep blood agar as de-

scribed previously (Murphy et al. 1995).

To determine DNA loss in the inactivated lkt operons from

M. ruminalis, we estimated the length of the region in six nonhe-

molytic strains using a two-part strategy. First, we searched for

conserved sequences in the 5¢ and 3¢ flanking regions of the lkt

operon between the genomes of the b-hemolytic strain HPA113

and the nonhemolytic strain HPA92T. We constructed a probe in

the 5¢ flanking region (hslU gene) of the lkt operon from the pub-

lished sequence of strain HPA113 (GenBank: AY425280) by using

the forward primer HPA113.1_UP (5¢-GCGAAAGATCAA

TGGGGTAA-3¢) in conjunction with the reverse primer

HPA113.1_DOWN (5¢-CACGAATCGGTAAACGACCT-3¢).The conditions were as for the lktA probe above, but with

annealing at 55�C. To construct phagemid libraries for strain

HPA92T, we partially digested genomic DNA with the restriction

enzyme Sau3AI. Aliquots were run on agarose gels and fragments

of approximately 3–6 kb were cloned into the BamHI site of the

Zap Express vector according to the manufacturer�s instructions

(Stratagene). The ligation mixture was packaged in vitro into the

Gigapack III Gold Packaging Extract and transfected into Esc-

herichia coli XL1-Blue MRF¢ according to the manufacturer�sinstructions (Stratagene). The plaques were lifted and cross-linked

to a 0.45-lm nitrocellulose membrane (Millipore) according to the

manufacturer�s instructions (Stratagene). The phagemid libraries

were screened for hslU clones and the pBK-CMV phagemid vector

was excised in vivo from the ZAP Express vector using the ExAssist

helper phage and E. coli XLOLR according to the manufacturer�sinstructions (Stratagene). The plasmids were then directly se-

quenced (GenBank AY425275).

To amplify the region in the nonhemolytic M. ruminalis strains,

we designed the forward primer rumpop_UP (5¢-AATGGTTGA

AGCGATGAAGG-3¢) and the reverse primer rumpop_DOWN

(5¢-TTGCGGTAGCCAAGAGAAAG-3¢) from conserved se-

quences in the 5¢ and 3¢ flanking regions between the genomes of

strains HPA113 and HPA92T. We used the Expand Long Template

PCR System according to the manufacturer�s instructions (Roche).

The cycling conditions were initial denaturation at 94�C followed

by 30 cycles at 94�C for 1 min, 52�C for 1 min, and 68�C for 8 min,

finishing with extension at 72�C for 10 min. We could not amplify

the region from the two nonhemolytic strains HPA81 and HPA93,

presumably because substitutions have resulted in imperfect mat-

ches of the primers.

Model Fit and Model Selection

The Akaike Information Criterion (AIC) was used to assess how

well various substitution and topology models fit the sequence data

(Akaike 1973; Burnham and Anderson 2002; Posada and Buckley

2004). The AIC is an estimate of the amount of information that is

lost when a given model is used to approximate the full truth (the

relative Kullback-Leibler distance). The AIC is a function of the

maximized log-likelihood (lnL) and the number of estimated

parameters (K) for a model: AIC = –2lnL + 2K, with lower AIC

values being better. From AIC, it is also possible to compute

Akaike weights (Burnham and Anderson 2002; Posada and

Buckley 2004). The Akaike weight of a model can be interpreted as

the conditional probability of the model given the data and the set

of initial models. It is possible to estimate the relative importance

of a model feature by simply summing Akaike weights across the

subset of models sharing that feature. Inference can thus be based

on a large set of models simultaneously (Burnham and Anderson

2002; Posada and Buckley 2004). Among other things, this is

helpful in avoiding the model selection problems associated with

misspecification (Zhang 1999). Here, AIC is used to select the best

model for construction of phylogenetic trees and for determining

whether there is support for a hard polytomy in M. ruminalis.

Reconstruction of Phylogenetic Trees

A species phylogeny was necessary to test the HGT of the lkt

operon. The relationships of the strains in this study were inferred

Fig. 1. Phylogenetic relationships,habitats, lktA genotypes of the fiveMannheimia subclades. Redrawn fromAngen et al. (1999).

425

Table 1. Strains used in this study

Taxona Strain ID Host Country

GenBank accession no.b

16S rRNA lktA (+845/+1302)

M. haemolytica (Mh)

Biogroup 1 PHL213 Ruminantia DQ301920 DQ301928

Biogroup 1 CCUG 12392T Ruminantia UK AF060699 DQ301929

M. glucosidal (Mgl)

Biogroup 3A P731 Ruminantia USA AF053888 DQ301930

Biogroup 3B P925T Ruminantia Scotland AF053889 DQ301931

Biogroup 3C UT18 Ruminantia Scotland AF053890 DQ301932

Biogroup 3C HPA117 Ruminantia Denmark

Biogroup 3D H62 Ruminantia Belgium DQ301921 DQ301933

Biogroup 3D H63 Ruminantia Belgium

Biogroup 3E P741 Ruminantia USA DQ301922 DQ301934

Biogroup 3F P933 Ruminantia Scotland DQ301923 DQ301935

Biogroup 3G P737 Ruminantia USA AF053891 DQ301936

Biogroup 3H P733 Ruminantia USA AF053892 DQ301937

Biogroup 9 P730 Ruminantia USA AF053897 DQ301938

Biogroup 9 228 Ruminantia Germany

M. granulomatis (Mgr)

Biogroup 3J W4672/1 Ruminantia Australia DQ301924 No product

Biogroup 3J 73992/T6c Ruminantia Denmark

Bt 20 biovar 1 P411 Leporidae Denmark

Bt 20 biovar 1 Ph13 Leporidae France AF053901 DQ301939

Bt 20 biovar 2 BJ1680.3 Leporidae Belgium DQ301925 DQ301940

[P.] granulomatis P1135/26T Ruminantia Brazil AF053902 DQ301941

[P.] granulomatis P1136/115 Ruminantia Brazil

[P.] granulomatis P1137/294 Ruminantia Brazil

[P.] granulomatis Carter 145/91 Ruminantia Brazil

[P.] granulomatis Carter 162/92 Ruminantia Brazil

[P.] granulomatis Carter 13B Ruminantia Brazil

M. varigena (Mv)

Biogroup 6 177T Ruminantia Germany AF053893 DQ301942

Biogroup 6 V1835 Ruminantia Australia AY425282 DQ301943

Bt 15 biovar 1 P655 Suidae Denmark AF053899 DQ301944

Bt 15 biovar 2 3997/82 Suidae Denmark DQ301926 DQ301945

Bt 36 H39 Ruminantia Belgium DQ301927 DQ301946

M. ruminalis (Mr)

Biogroup 1 UT26 Ruminantia Scotland AF053887 DQ301947

Biogroup 8D HPA98 Ruminantia Scotland AF053896

Biogroup 10 HPA95 Ruminantia Scotland AY425289 DQ301948

Biogroup 10 HPA114 Ruminantia Scotland AY425290

Biogroup 10 UT27 Ruminantia Scotland AY425291

Bt 18 biovar 1 HPA92T Ruminantia Scotland AF053900

Bt 18 biovar 1 HPA81 Ruminantia UK U57077

Bt 18 biovar 2 HPA113 Ruminantia UK AY425283 DQ301949

Bt 18 biovar 2 HPA90 Ruminantia AY425284

Bt 18 biovar 2 UT38 Ruminantia AY425285

Bt 18 biovar 3 HPA109 Ruminantia UK AY425286



Unclassified strains (Mx)

Biogroup 7 R19.2 Ruminantia Scotland AF053894

Biogroup 8Ac HPA102 Ruminantia Scotland AF053895

Biogroup 8B 274 Ruminantia Germany AY425292

Biogroup 8B R108B(3) Ruminantia Scotland AY425293

Biogroup 8C M14.4 Ruminantia Scotland AY425294

Biogroup 10 BJ3956.1 Ruminantia Belgium AY425295

Biogroup 10 HPA121 Ruminantia Scotland AF053898

Bt 39 BNO311 Ruminantia Australia AF216870 DQ301950

Bt 39 BNO423 Ruminantia Australia





(Continued)

426

with 16S rRNA; these sequences have been used successfully for

systematic studies in this group (Angen et al. 1999). Sequences for

19 strains were obtained from GenBank. The primers previously

described by Angen et al. (1999) were used to amplify and directly

sequence 16S rRNA from 22 additional strains. The 16S rRNA

sequences were aligned using Dialign 2 (Morgenstern 1999) with

default settings. Phylogenetic trees were reconstructed using

Bayesian techniques as implemented in the program MrBayes

version 3.1.1 (Huelsenbeck and Ronquist 2001; Ronquist and

Huelsenbeck 2003). The best-fitting model was GTR + invgamma

based on the AIC computed by the program MrModeltest version

2.2 (Nylander 2004). Markov chain Monte Carlo (MCMC) was

run for 10 million generations with four chains while sampling once

every 100 generations. Convergence was confirmed by comparing

the results of two independent runs. The program Tracer version

1.3 (Rambaut and Drummond 2004) was used to determine burn-

in and also for further confirmation of proper mixing and adequate

run-length. A burn-in of 1 million generations (10,000 samples) was

used. The distribution of 16S rRNA trees resulting from MCMC

was summarized in the form of a consensus tree with all compatible

bipartitions included by using the authors� own software in a

manner that is essentially identical to what is obtained when using

MrBayes� sumt command with the setting contype=allcompat,

except that branch lengths were averaged over all trees, setting the

branch length to zero for those trees that did not contain the

corresponding bipartition as suggested by Felsenstein (2003). The

tree was rooted based on a maximum likelihood analysis reported

by Angen et al. (1999).

To test for the origin of the lkt operons, we inferred the rela-

tionships of 101 strains with partial sequences from genes encoding

cytotoxic RTX proteins. The +845/+1302 region of lktA from 23

lktA {+} strains was amplified by using the PCR primers man-

pop_UP and manpop_DOWN. The reaction and cycling condi-

tions were as above. These PCR products were then directly

sequenced. We could not amplify lktA from the lktA {+} strain

W4672/1, presumably because substitutions have resulted in

imperfect matches of the primers. We used the +845/+1302 region

of lktA from M. haemolytica strain PHL213 for selecting putative

orthologues from the NCBI nonredundant (nr) database by per-

forming TBLASTX searches (Altschul et al. 1990). We identified 78

sequences corresponding to genes encoding cytotoxic RTX proteins

(E-value < e)10; score > 99) (see Table 2 for a complete list of

retrieved rtxA nucleotide sequences, along with their accession

numbers). Phylogenetic trees of the nucleotide sequences were

reconstructed in the same way as the 16S rRNA trees. The distri-

bution of rtxA trees from MCMC was summarized in the form of a

50% majority rule consensus tree corresponding to MrBayes� sumt

command with the setting contype=halfcompat. The tree was

rooted by using the eight Bordetella sequences as outgroup.

Hard Versus Soft Polytomy

To test for the presence of hard polytomies in M. ruminalis, we

explicitly assessed support for a range of alternative tree topologies.

There are 11 internal branches in M. ruminalis, 8 of which have

posterior probabilities below 95%. Using software written by us, we

constructed the 256 possible variants, where one or more of these

eight branches have been collapsed. To each of these alternative

trees, we then fitted various substitution models using the program

baseml from the PAML package version 3.14 (Yang 1997). The

models we used were GTR+ gamma and four different versions of

nparK = 2. The nparK = 2 model accounts for different rates

across sites by having several separate rate categories, each

applying to a different proportion of sites (Yang and Roberts

1995). Here, we tested versions with two, three, four, or five rate

categories. We fitted the different models to each tree topology, in

each case recording the number of free parameters (including

parameters associated with branch lengths) and the maximized

likelihood. To check for convergence and ensure that the global

maximum had been found, we fitted each model to each topology

in five independent runs. For each model/topology combination,

we used the highest of the five independently obtained likelihoods

for further computations. This allowed us to calculate AIC and

Akaike weights, and from this we could finally compute the cross-

model support for each individual tree topology. The main purpose

of fitting a range of models was to avoid the errors associated with

misspecification (Zhang 1999).

We believe that our strategy holds some advantages over pre-

vious methods which were based on classical hypothesis testing

(Jackman et al. 1999; Slowinski 2001). The main problem with such

approaches is that we are interested in assessing the support for the

null hypothesis (polytomy) and this information is not contained in

the p value because failure to reject a null model does not neces-

sarily mean that it is well supported. This problem is exacerbated

when several internal branches are tested simultaneously and it is

problematic how (and whether) to correct for multiple testing

artifacts. Thus, conservative correction methods, such as Bonfer-

roni, will result in a bias for the polytomy-containing topologies

(Slowinski 2001).

Compositional Analysis

To rank genes according to their convergence to mutational bias in

the genome ofM. haemolytica, we retrieved nucleotide sequences of

genes from the NCBI nonredundant (nr) database. When the

source of a gene was not clearly defined, these sequences were

excluded from the present study. When multiple alleles of the same

gene were retrieved, we used those sequence data as queries to

search the preliminary M. haemolytica strain PHL213 genome by

using BLASTN (Altschul et al. 1990) with default settings, and

only the allele with the highest E-value was included in the present

study. In total, we found 56 genes (�68 kb, corresponding to

�2.8% of the genome size) using this procedure (see Table 3 for a

complete list of retrieved M. haemolytica genes, along with their

accession numbers). We used this data set to create an average

genome signature (l) based on the relative 3:1 dinucleotide bias (z)

in individual genes as described previously (Hooper and Berg

2002). This method uses a variant of Hotelling�s T2 statistics as a

Table 1. Continued

Taxona Strain ID Host Country

GenBank accession no.b

16S rRNA lktA (+845/+1302)



aBt, Bisgaard taxon.bSequences in italics have been published previously.cStrains belonging to biogroup 8A groupwithM. glucosida based onmaximum likelihood analysis of 16S rRNA sequences (Angen et al. 1999).

427

multivariate distance measure between each gene to the average

genome signature and identifies deviant genes at a given signifi-

cance level.

Sequence Data

Sequences have been deposited in the GenBank database (accession

numbers AY425275, AY425282–AY425295, and DQ301920–

DQ301950).

Results

3:1 Dinucleotide Bias of the lkt Genes inM. haemolytica

Employing a compositional approach to the identi-fication of horizontally transferred genes requiresthat genes from the donor and recipient have a dif-ferent nucleotide composition. Relative abundancesin dinucleotide frequencies are unique to most livingorganisms and consequently constitute a genome

signature that is discriminatory between sequencesfrom different organisms (Karlin et al. 1997;Campbell et al. 1999). From these signatures, thedistance of an individual gene to the average values ofthe genome can be calculated. Recent work byHooper and Berg (2002) redefined this approach byusing only the average relative 3:1 dinucleotideabundance, which is the combination that is leastaffected by codon and amino acid usage. Here weused a data set of 56 genes to create an averagegenome signature (l) of M. haemolytica based on therelative 3:1 dinucleotide bias (z) in individual genes.The multivariate distance measure (T2) between eachgene to the average genome signature was calculated(Table 3). The T2 values were F-distributed with(15,41) degrees of freedom, corresponding to a criti-cal limit of T2 � 33 (a = 0.1). This means that themethod is expected to pick out at least 10% (five orsix) of the genes that deviate most from the genomicsignature because the significance level is lower inbiological data sets (Hooper and Berg 2002). The T2

Table 2. List of retrieved rtxA nucleotide sequences

Genea Taxon GenBank accession no.

lktA Mannheimia

M. haemolytica (Mh) M20730, M24197, AF314514, AF314503, AF314507, AF314505, AF314504,

AF314506, AF314512, AF314510, AF314509, AF314508, AF314513, AF314511,

AF414141, AF314515, AF314516

M. glucosida (Mgl) U01215, AF314519, AF314522, AF314518, AF314521, AF314517, AF314520

M. cf. haemolytica (Mh-like) L12148

Pasteurella

[P.] trehalosi (Pt) U01216, Z26247, AF314526, AF314525, AF314524, AF314523

Actinobacillus

apxIIA A. porcitonsillarum (Apo) AY795600

A. pleuropneumoniae (App) M30602, AY736188, X61111, AY232288, AF363362

A. suis (As) M90440

apxIIIA Actinobacillus

A. pleuropneumoniae (App) L12145, X80055, X68815, AF363363

apxIA Actinobacillus

A. pleuropneumoniae (App) X73117, D16582, U04954, X52899, U05042, AF240779, X68595, AF363361

aqxA Actinobacillus

A. cf. equuli (Ae-like) AF381184

A. equuli (Ae) AF381185

paxA Pasteurella

P. aerogenes (Pa) U66588

hlyA Escherichia

E. coli (Ec) AY258503, AB011549, AF074613, AF043471, AB032930, X79839, X94129 M10133,

AE016766, AJ488511, AJ494981, U12572, M14107, X86087

ltxA Actinobacillus

A. actinomycetemcomitans (Aa) X16829, M27399

mbxA Moraxella

M. bovis (Mb) AF205359

cyaA Bordetella

B. hinzii (Bh) DQ102773, DQ007078

B. parapertussis (Bpa) AJ249835, BX640423

B. pertussis (Bpe) BX640413, Y00545

B. bronchiseptica (Bb) BX640437, Z37112

aGene names are those reported in protein databases or have been assigned by us on the basis of orthology relationships. The genes and their

accession numbers are listed in the subfamily in which they are included.

428

values for the lkt genes ranged from 11.22 to 16.07,suggesting that they are nondeviant from the averagegenome signature of M. haemolytica.

Phylogeny of 16S rRNA Sequences

The 16S rRNA data set included 1257 bp afterremoval of ambiguous bases. Both runs convergedand the consensus tree with all compatible biparti-tions included and with branch lengths averaged overall trees, setting the branch length to zero for thosetrees that did not contain the corresponding biparti-tion, is shown in Fig. 2, along with posterior proba-bilities (PPs). This phylogram is largely topologicallyconcordant with the maximum likelihood analysisreported by Angen et al. (1999) and indicates mod-erate to strong PP support for monophyly of thefive subclades (M. haemolytica + M. glucosida,M. ruminalis, M. granulomatis, M. varigena, andsubclade V comprising unclassified strains) (PP =82%–100%) and bifurcation order among any subc-lades (PP = 100%) but very low PP support forbifurcation order among strains within M. ruminalis.There was strong PP support for a sister-group rela-tionship between M. haemolytica + M. glucosida andM. ruminalis as expected (PP = 100%).

Hard Versus Soft Polytomy for M. ruminalis

Our Bayesian analyses showed that most internalbranches are very short and indicated low PP supportfor bifurcation order among strains within M. rumi-nalis (Fig. 2), suggesting simultaneous divergence(hard polytomy) or at least very rapid early diver-gence (soft polytomy). The hypothesis that theM. ruminalis subclade contains hard polytomies wasapproached as a model selection problem. First, weconstructed the full set of 256 tree topologies whereone or more of the eight internal branches with PPsbelow 95% have been collapsed. Then we used themaximum likelihood principle to fit five differentsubstitution models to each of these alternative treetopologies. Lastly, we calculated the AIC and Akaikeweights, and from this we could finally compute thecross-model support for each individual tree topol-ogy. The tree topology with the strongest cross-modelsupport (0.225) suggests collapse of seven of the eightinternal branches with PPs <95% (Fig. 3). Thetopology (HPA113 + HPA98) is concordant with theBayesian analysis (PP = 82%). The next three treetopologies ranked according to their cross-modelsupport (0.081–0.083) all have one less collapsedinternal branch (data not shown).

Distribution of the lktA Genotype and Phenotypeon the Mannheimia Phylogeny

If the ancestor of M. haemolytica + M. glucosida, orother major subclades, gained the lkt operon viaHGT, we would expect that the lktA genotype and

Table 3. List of retrieved M. haemolytica genes ranked accordingto their multivariate distance measures to the average genomesignature

Gene GenBank accession no. Length T2

wecB AF170495 1272 2.58

Gale U39043 1017 3.67

tfbA U73302 1755 5.99

Gcp U15958 978 6.23

phyA AF170495 2091 6.89

aroA U03068 1305 7.72

exbD U62565 438 8.08

exbB U62565 459 9.24

recA AF176376 1107 9.46

Rnt U73302 651 9.61

Crp L47536 675 9.90

Soda L47537 642 10.75

lktD M24197 1437 11.22

pomA AF133259 1137 11.31

tbpA U73302 2793 11.51

wecC AF170495 1119 11.75

lktB M24197 2127 12.09

lapT M59210 714 12.18

plpA L11037 834 12.56

Res AF060119 2640 13.17

lktC M24197 504 13.33

fbpC AF047427 1011 13.39

dnaK AF017730 1896 13.41

plpB L11037 831 13.44

hsdS U46781 1329 13.68

ssa1 M62363 2799 13.99

cpxC AF170495 1104 14.13

potD U25682 1095 14.14

cpxA AF170495 648 14.44

lapA M59210 1323 14.86

envM AF033119 456 14.91

plpC L11037 792 15.11

fnrP AF033119 774 15.16

plpD AF058703 855 15.47

Mod AF060119 2121 15.95

lktA M24197 2862 16.07

Irp AY028475 2301 16.24

orf 5¢ (mpa1) S68137 633 16.27

mpa1 S68137 1179 16.58

Rph AY028475 717 17.02

kdsA U52971 855 17.02

cpxB AF170495 798 17.49

fbpA AF047427 1029 17.63

dnaJ AF017730 1140 17.63

tonB U62565 741 17.96

lapB M59210 678 19.48

fbpB AF047427 1578 19.88

lppC U42028 1641 19.89

cpxD AF170495 1185 20.23

ihfA U56138 300 20.78

lapC M59210 522 21.36

lpsA U15958 792 23.64

hsdR U46781 3168 25.86

Fis U73302 297 26.97

plpE AF059036 1071 28.87

alxA–hsdM U46781 1851 44.54

429

the corresponding b-hemolytic phenotype are presentonly in these subclades. Alternatively, finding thatthese character states are present in different majorsubclades would suggest vertical descent, accompa-nied by major divergences in the lktA genotype andthe corresponding b-hemolytic phenotype. The lktAgenotype and the corresponding b-hemolytic pheno-type are given in Fig. 2 and Table 4. Both characterstates were present among any subclades, includingall strains belonging to M. haemolytica + M. glu-cosida (15 strains), M. granulomatis (11 strains), andM. varigena (5 strains), but only a fraction of strainsbelonging to M. ruminalis (7 of 13 strains) andsubclade V (13 of 14 strains). All lktA {+} strainswere also b-hemolytic, whereas all lktA {)} strainswere nonhemolytic.

Determination of DNA Loss in the Inactivated lktOperons from M. ruminalis

We used a two-part strategy to determine DNA lossin the inactivated lkt operons from M. ruminalis.First, we searched for conserved sequences in the 5¢and 3¢ flanking regions of the lkt operon between thegenome of the b-hemolytic strain HPA113 and thatof the nonhemolytic strain HPA92T and found thatthe hslU-lapB sequence in the 5¢ flanking region andthe tauB sequence in the 3¢ flanking region of the lktoperon from strain HPA113 were conserved in thesequence hslU-lapB-tauB of strain HPA92T. Then wedesigned forward and reverse primers in these con-served sequences, allowing us to determine DNA lossin the inactivated lkt operons from nonhemolytic

Fig. 2. Distribution of16S rRNA trees fromMCMC summarized in theform of a consensus treewith all compatiblebipartitions included andwith branch lengthsaveraged over all trees,setting the branch length tozero for those trees that didnot contain thecorresponding bipartition.The tree was rooted basedon a maximum likelihoodanalysis reported by Angenet al. (1999). Posteriorprobability values areshown for all compatiblebipartitions. Presence/absence of the lktAgenotype and thecorresponding b-hemolyticphenotype is indicated by{+/–;+/–}. Monophyly ofthe five subclades reportedby Angen et al. (1999) isindicated by black circles.Strains present in the lktAtree (Fig. 4) are indicatedby asterisks.Nomenclature: sequencenames containabbreviations of thetaxonomic group (genusand species) followed bythe corresponding strainID as listed in Table 1.

430

strains (measured as the reduction in amplicon size).The amplicon sizes are given in Table 5, along withtheir last b-hemolytic ancestor. The amplicon sizesfrom three of four nonhemolytic strains, includingstrain HPA92T, were 0.5 kb, corresponding toabsence of the entire lkt operon. The amplicon sizefrom the nonhemolytic strain HPA88, along with theb-hemolytic strain HPA113, was 7.9 kb, corre-sponding to the presence of the entire lkt operon.

Phylogeny of the rtxA Sequences

The distribution of the lkt operon can always beexplained by vertical inheritance followed by losses incertain subclades. Phylogenetic reconstructions aretherefore necessary to distinguish between this sce-nario and a scenario whereby the ancestor ofM. haemolytica + M. glucosida, or other major subc-lades, gained the lkt operon via HGT. Loss of the lktoperon is expected to produce phylogenetic treesthat broadly resemble the accepted monophyletic

organismal groups, whereas HGT is expected to pro-duce trees where the rtxA sequences from the recipientsubclades group with homologous sequences fromtheir donors. The rtxA dataset included 458 bp afterremoval of ambiguous bases. Both runs converged andthe 50% majority rule consensus tree is shown inFig. 4, along with PPs. There was strong support forgrouping of the sequences from the Mannheimia and[P.] trehalosi strains (PP = 100%). There was alsostrong support for grouping most of the sequencesfromM. haemolytica+M. glucosida andM. ruminalis(exceptM. glucosida strain P730) and all the sequencesfrom [P.] trehalosi (PP = 100%). Support for bifur-cation order of the [P.] trehalosi group and strainBNO311 belonging to subclade V was low (PP =62%). Thismeans that the [P.] trehalosi sequences forma sister group to most of the sequences fromM. haemolytica + M. glucosida and M. ruminalis(exceptM. glucosida strain P730) and, possibly, strainBNO311.

Discussion

Vertical Inheritance

Prior to this work, no Mannheimia rtxA sequencesfrom outside M. haemolytica + M. glucosida hadbeen reported and their arrival in this subclade wasunclear. Davies et al. (2002) used phylogeneticmethods to compare 6952 bp of the lkt operon fromM. haemolytica, M. glucosida biogroup 3B, and [P.]trehalosi and noted that the ancestral bovine operonfrom M. haemolytica has been acquired by HGTfrom a more distantly related donor. However, sucharguments based on a relatively small number of taxacan be misleading because of problems in informa-tion content. Our analyses, based on a larger collec-tion of strains and utilizing a combination ofcompositional and phylogenetic methods, indicatevertical inheritance from the last common ancestor ofgenus Mannheimia.

Given the collection of strains, information ontheir lktA genotype and the corresponding b-hemo-lytic phenotype, and an organismal phylogeny thatdescribes bifurcation order, the characteristics oftheir ancestors can be inferred. Our analyses revealedthat both character states were present amongany subclades, including all strains belonging toM. haemolytica + M. glucosida, M. granulomatis,and M. varigena (Fig. 2 and Table 4). These resultsare compatible with a history of vertical inheritanceof the lkt operon from the last common ancestor ofgenus Mannheimia to any of its descendants followedby losses in the terminal branches ofM. ruminalis andsubclade V, thus opposing the hypothesis that theancestor of M. haemolytica + M. glucosida gainedthe lkt operon via HGT.

Table 4. Distribution of the lktA genotype revealed by Southernblot and the corresponding b-hemolytic phenotype among strainsused in this study

TaxonaNo. of

strains

lktA

genotype

b-hemolytic

phenotype

M. haemolytica

Biogroup 1 2 + +

M. glucosidal

Biogroup 3A-H 10 + +

Biogroup 9 2 + +

M. granulomatis

Biogroup 3J 2 + +

Bt 20 biovar 1 2 + +


[P.] granulomatis 6 + +

M. varigena

Biogroup 6 2 + +



Bt 36 1 + +

M. ruminalis

Biogroup 1 1 + +

Biogroup 8D 1 ) )Biogroup 10 3 + +

Bt 18 biovar 1 2 ) )Bt 18 biovar 2 3 + +

Bt 18 biovar 3 2 ) )Bt 18 biovar 4 1 ) )

Unclassified strains

Biogroup 7 1 + +

Biogroup 8A-Cb 4 + +

Biogroup 10 (BJ3956.1) 1 + +

Biogroup 10 (HPA121) 1 ) )Bt 39 8 + +

aBt, Bisgaard taxon.bStrains belonging to biogroup 8A group with M. glucosida based

on maximum likelihood analysis of 16S rRNA sequences (Angen

et al. 1999).

431

Reconstruction of the rtxA tree provided strongsupport for grouping all of the sequences fromMannheimia, although the analysis also revealed anumber of conflicting phylogenetic signals (Fig. 4).This incongruence between gene trees and organismalphylogenies could arise from phylogenetic noise dueto convergence and/or sequences with poor phylo-genetic signal or from gene transfer events (homolo-gous recombination or HGT). The strong support fora sister-group relationship between the [P.] trehalosisequences and most of the sequences from M. haem-olytica + M. glucosida and M. ruminalis (exceptM. glucosida strain P730) is interesting because itsuggests a history of gene transfer between theancestor of M. haemolytica + M. glucosida andM. ruminalis and [P.] trehalosi. The lkt operon fromthe ancestor of these Mannheimia subclades does notseem to have been acquired by HGT from [P.] tre-halosi, since this scenario would require loss of theoperon prior to the gene transfer event, somethingthat seems unlikely given the presence of the lktAgenotype and the corresponding b-hemolytic pheno-type among any subclades within genus Mannheimia.Therefore, the incongruence observed in the rtxA treeis compatible only with a history of HGT from theancestor of M. haemolytica + M. glucosida andM. ruminalis to [P.] trehalosi or homologousreplacement of vertically inherited +845/+1302regions. Previous analyses of the lkt genes fromM. haemolytica, M. glucosida biogroup 3B, and [P.]trehalosi have revealed the relevance of gene transferin shaping the lkt operons (Davies et al. 2001, 2002).Although this work suggested a mosaic origin of thelktBD genes from [P.] trehalosi, the +845/+1302region of lktA from [P.] trehalosi showed no evidenceof recent gene transfer (Davies et al. 2001, 2002).From a phylogenetic perspective, these results con-cord with the relatively old age of this gene transferevent followed by divergent evolution of the +845/+1302 region of lktA in [P.] trehalosi. In order todetermine whether the incongruence observed in thertxA tree is the result of HGT from the ancestor ofM. haemolytica + M. glucosida and M. ruminalis to[P.] trehalosi or homologous replacement of vertically

inherited +845/+1302 regions, we need to addressthe question of the origin of the lkt operon from [P.]trehalosi. However, the unstable position of genusMannheimia and [P.] trehalosi in the Pasteurellaceaetree (Christensen et al. 2004) and the lack of a com-positional signature for [P.] trehalosi limit our abilityto evaluate these alternative scenarios.

The results of Davies et al. (2002) also revealedthat a 4.4-kb region of the ancestral bovine operonfrom M. haemolytica strains belonging to the bovineA2 complex, including the entire lktA gene (allelegroup 2), was more divergent than the homologoussequences from other strains belonging to M. haem-olytica, M. glucosida biogroup 3B, and [P.] trehalosi,suggesting that the entire lkt operon has been ac-quired by HGT from a more distantly related donor.Following Davies and coworkers� hypothesis, wewould expect a robust and systematic incongruencetoward the donor. However, we found strong supportfor grouping most of the rtxA sequences fromM. haemolytica + M. glucosida and M. ruminalis(except M. glucosida strain P730), including thosesequences belonging to allele group 2 (Fig. 4).

The multivariate distance measure (T2) betweeneach gene to the average genome signature (l) ofM. haemolytica based on the relative 3:1 dinucleotidebias (z) in individual genes identified the lkt genes asnondeviant. These results also support a historyof vertical inheritance of the lkt operon from thelast common ancestor of genus Mannheimia toM. haemolytica, thus opposing the hypothesis thatthe ancestor of M. haemolytica + M. glucosidagained the lkt operon via HGT. However, false neg-atives (missed transferred genes) arise when geneshave ameliorated due to the mutational processesaffecting the recipient genome or the genes are closelyrelated to the recipient genome in terms of contextbias (Lawrence and Ochman 1997). Therefore, theobserved distances between each lkt gene to theaverage genome signature cannot rule out ancientHGT events, although this is an unlikely scenario,since the data from both phylogenetic methods sup-port a history of vertical inheritance from the lastcommon ancestor of genus Mannheimia.

Table 5. Amplicon sizes from nonhemolytic M. ruminalis strains, along with their last b-hemolytic ancestors

Strain ID lktA genotype b-hemolytic phenotype Last b-hemolytic ancestora Amplicon size (kb) Size of deletion (kb)

HPA98 – – HPA113 + HPA98 �0.5 �7.4

HPA92T – – LCA �0.5 �7.4

HPA81 – – LCA No product ?

HPA109 – – HPA114 + HPA109 �0.5 �7.4

HPA93 – – LCA No product ?

HPA88 – – LCA �7.9 �0.0

aLCA, Last Common Ancestor of M. ruminalis.

432

Radiation in M. ruminalis

In order to explain the discontinuous distribution ofthe lktA genotype and the corresponding b-hemolyticphenotype in M. ruminalis, it was necessary to resolvethe bifurcation order among strains within thissubclade. Our expanded analyses allowed us todemonstrate that two hard polytomies are implicated(Fig. 3). The size of the early radiation (measured asthe number of new genotypes) is eight, suggestingthat divergence following colonization of the ovinerumen was very rapid.

Radiation of a single lineage into a range ofgenotypes, as appeared to happen once M. ruminaliscolonized the rumen, is not surprising given thenumber of new niches that would be encountered.However, the results presented here do not allow usto test whether radiation reflects the differentiation ofa single ancestor into an array of lineages that inhabita variety of environments and that differ in themorphological and physiological traits used to ex-ploit those environments (adaptive radiation) or ra-pid proliferation accompanied by negligible orinfrequent ecological differentiation (nonadaptiveradiation). There is some evidence that strainsbelonging toM. ruminalis differ in physiological traits(Angen et al. 1997b), but a fit between the diversephenotypes of descendant lineages and their diver-gent environments and evidence that those pheno-types are indeed useful where they are employed are

needed to fulfill the criteria of adaptive radiation asproposed by Schluter (2000).

Mechanisms of Gene Loss in M. ruminalis

Radiation is consistent with losses of the lkt operonsin M. ruminalis. If one assumes that the evolvingpopulations adapted to different ecological nichesoffering different conditions of growth, then thisdiversity would imply that different pools of unusedgene activities have been lost in those populations.

Genes and gene activities are generally thought tobe lost from populations over the course of evolutionby one of two evolutionary mechanisms: (i) antago-nistic pleiotropy; or (ii) mutation accumulation. Un-der antagonistic pleiotropy, adaptation to theselective environment and functional decay in otherenvironments are caused by the same mutations.Independent of adaptive mutations, genes can also belost from a population by the fixation of neutral ornearly neutral mutations via selection-indepen-dent genetic drift. This mechanism is thought toaccount for much of the loss of genes in prokaryotes(Lawrence and Roth 1999; Mira et al. 2001).

These two processes have long been recognized, buttheir relative contributions are not easy to examine.Our data showed that loss of the b-hemolytic pheno-type has occurred in four of eight early radiating lin-eages (Fig. 3), accounting for 67% of all losses inM. ruminalis. This pattern is consistent with antago-nistic pleiotropy, which predicts that most lossesshould occur early when adaptation to the rumen isfastest and that most lineages should exhibit parallelloss of functions (Cooper and Lenski 2000).We do notexpect this association between the dynamics ofadaptation and loss via mutation accumulation, whichpredicts that losses of unused functions should accu-mulate stochastically at a constant rate that dependsonly on themutation rate in the relevant genes (Cooperand Lenski 2000). Our results therefore suggest thatinactivation of the lkt operons in the radiating lineagesof M. ruminalis have catalyzed their adaptation to acommensal environment and reproductive isolation(speciation), although they do not allow us to experi-mentally verify that functional decay of the lkt operonsis beneficial in the ruminal environment.

The loss of gene content of bacterial species isthought to occur by the same molecular mechanismsthat influence their ability to incorporate DNA fromother species: (i) RecA-dependent homologousrecombination; or (ii) illegitimate recombination.Because the frequency of homologous recombinationdoes not appear to be constrained by the distancebetween the repeated sequences, it often results inlarge deletions (Moran and Mira 2001). On the con-trary, illegitimate recombination between closelyspaced repeats results in multistep deletion of pseud-

Fig. 3. Cladogram showing the M. ruminalis tree topology withthe strongest cross-model support. The tree was rooted in the sameway as in Fig. 2. Posterior probability values are shown for allcompatible bipartitions. Nodes with posterior probabilities above95% were excluded from the analysis and are indicated by blackcircles. Nodes with posterior probabilities <95% that were notcollapsed by the analysis are indicated by white circles. Hardpolytomies are indicated by numbers, suggesting early (1) and late(2) radiation. Presence/absence of the lktA genotype and the cor-responding b-hemolytic phenotype is indicated by {+/–;+/–}.

433

ogenes (Andersson and Andersson 2001; Silva et al.2001). The results of Andersson and Andersson (2001)revealed that the mean and median sizes of the dele-

tions in pseudogenes from four Rickettsia genomeswere 51.2 and 4 bp per event, respectively, althoughtwo large deletions, of 599 and 767 bp, were observed.

Fig. 4. Distribution of rtxA trees fromMCMC summarized in theform of a 50% majority rule consensus tree. The tree was rooted byusing the eight Bordetella sequences as outgroup. Posterior prob-ability values are shown for all compatible bipartitions. Groupingof the sequences from the Mannheimia and [P.] trehalosi strains isindicated by a black circle. The sister-group relationship betweenthe [P.] trehalosi sequences and most of the sequences from M.haemolytica + M. glucosida and M. ruminalis (except Mgl_P730) is

indicated by a white circle. Grouping of most of the sequencesfrom M. haemolytica + M. glucosida and M. ruminalis (exceptMgl_P730), including those sequences belonging to allele group 2,is indicated by a gray circle. Nomenclature: sequence names con-tain abbreviations of the taxonomic group (genus and species)followed by the corresponding strain ID and/or the accessionnumber as listed in Tables 1 and 2.

434

However, recent work has shed light on the role ofRecA-independent recombination events in fastreductive evolution. Nilsson and coworkers (2005)examined the rate and molecular mechanisms ofgenome reduction in Salmonella enterica by using se-rial passage and genetic selections. Those authorsshowed that the individual chromosomal deletionsvaried in size from�1200 to 202,232 bp. The potentialhomology at most of the deletion endpoints was £ 12bp, suggesting that rapid deletion of large block ofDNA, including functional genes or operons, couldarise from RecA-independent mechanisms.

We identified chromosomal deletions of �7400 bp,corresponding to the entire lkt operon, in three of fournonhemolytic strains, including strains HPA98 andHPA109, which diverged only recently from their lastb-hemolytic ancestors, whereas we found no evidencefor DNA loss in the inactivated lkt operon from strainHPA88 (Fig. 3 and Table 5). The results from strainsHPA92T and HPA88, which diverged from the lastcommon ancestor of M. ruminalis, do not allow usto examine DNA loss rates because of problemsassociated with estimating the age of initial deletionformation. However, following our hypothesis ofantagonistic pleiotropy, which predicts that functionaldecay of most of the lkt operons should occur earlywhen adaptation to the rumen is fastest, it is expectedthat similar DNA loss rates would result in equal sizesof deletions. Our results therefore support a very lowDNA loss rate in strain HPA88, although they do notrule out the alternative scenario of equal DNA lossrates, which implies that inactivation of the lkt operonfrom strain HPA88 occurred more recently comparedto other nonhemolytic strains. This potential discrep-ancy between the time of divergence from their lastb-hemolytic ancestors and the observed sizes of thedeletions points toward two most probable explana-tions. On one hand, it is possible that DNA loss occursat different rates in the radiating lineages of M. rumi-nalis due to differences in the molecular mechanismsinvolved such as those discussed above. On the otherhand, the results of Nilsson et al. (2005) showed thatthe DNA loss rate of WT bacterium was 0.05 bp perchromosome per generation and �50-fold higher in amutS mutant. These data suggest that high DNA lossrates could arise from defects in the methyl-directedmismatch repair (MMR) system. Although there issome evidence that strains belonging to M. ruminalisdiffer in DNA loss rates, we need to explore the dele-tion endpoints for repeats and the MMR system fordefects in order to estimate the molecular mechanismsresponsible for reductive evolution.

Implications for Pathogenic Adaptation and Speciation

The data collected here provide strong supportfor vertical inheritance of the lkt operon but do not

resolve its role in pathogenic adaptation and specia-tion of the M. haemolytica + M. glucosida subclade.Although genes and accessory elements gained fromdistantly related donors are responsible for manyinteresting adaptations of bacteria, other evolutionaryprocesses are by no means inconsequential for adap-tation to new ecological niches: (i) fixation of favor-able mutations via selective sweeps or populationbottlenecks; (ii) homologous replacement of allelesthat have evolved in ecologically distant donors; or(iii) genome rearrangements (deletions, duplications,translocations, and inversions) via homologousrecombination between direct and inverted repeats.

Previous works have revealed the relevance ofhomologous recombination between inter- and in-tragenera species in shaping the lkt operon (Davieset al. 2001, 2002). Our results are compatible with thepredictions of Davies et al. (2001, 2002) on themosaic origin of the lkt operon from M. haemolytica,M. glucosida biogroup 3B, and [P.] trehalosi, thusreaffirming the hypothesis that these gene transferevents could have important implications for patho-genic adaptation and speciation of the recipients.Indeed we have found a number of strains to be inincongruent positions on the rtxA tree (e.g., M. vari-gena strains 177T, V1835, and H39 group withM. glucosida strain P730 and M. granulomatis strainPh13). Since these potential gene transfer events donot result in an incongruent position of the involvedMannheimia strains out from this genus, it seemsreasonable to assume that they have occurred amongstrains belonging to genus Mannheimia via homolo-gous replacement of vertically inherited +845/+1302regions and not with other more distantly relatedtaxa. However, our rtxA data set only included 458bp after removal of ambiguous bases. In conse-quence, we need to address directly the question ofthe origin of each region by extending the data set ofDavies et al. (2002) with genes from more distantlyrelated taxa in order to explain the evolution of theentire lkt operon in light of homologous replacementof sequences outside the +845/+1322 region.

Conclusions

After the radiation from their common ancestor, theMannheimia subclades have taken divergent pathstoward their distinct lifestyles. However, we found noevidence for HGT of the lkt operon into any Mann-heimia subclades, suggesting that alternative pro-cesses are involved in pathogenic adaptation andspeciation. By contrast, the results presented heresuggest that losses of the lkt operons in the radiatinglineages of M. ruminalis have catalyzed their adap-tation to a commensal environment and reproductiveisolation (speciation).

435

Acknowledgments. We thank Christel G. Buerholt and Tony

Bønnelycke for technical assistance and Sean D. Hooper for

enlightening discussions. This work was supported by Grant

9702797 from the Danish Agricultural and Veterinary Research

Council.

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Evidence for Vertical Inheritance and Loss of the Leukotoxin Operon in Genus Mannheimia

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