Journal of Bacteriology, March 2004, p . 1484-1492, Vol . 186, No . 5

Bordetella Species Are Distinguished by Patterns of Substantial Gene Loss and Host Adaptation

C . A . Cummings,^1,2, M . M . Brinig,^1,2, P . W . Lepp,^1,2 S . van de Pas,^1,2, and D . A . Relman^1,2,3*

Departments of Microbiology and Immunology,¹ Medicine, Stanford University School of Medicine, Stanford, California 94305,² VA Palo Alto Health Care System, Palo Alto, California 94304³

Received 12 August 2003/ Accepted 10 November 2003

 
Pathogens of the bacterial genus Bordetella cause respiratory disease in humans and animals . Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified . To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysisof genome content in 42 Bordetella strains by hybridizationof genomic DNA to a microarray representing the genomes of three Bordetella species and by subtractive hybridization . Here weshow that B . pertussis and B . parapertussis are predominantly differentiated from B . bronchiseptica by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen,which may be important determinants of host specificity . Thespecies also exhibit sequence diversity at a number of surfaceprotein-encoding loci, including the fimbrial major subunitgene, fim2 . Gene loss, rather than gene acquisition, accompaniedby the proliferation of transposons, has played a fundamentalrole in the evolution of the pathogenic bordetellae and mayrepresent a conserved evolutionary mechanism among other groupsof microbial pathogens.

 
Bacteria of the genus Bordetella are important pathogens that cause respiratory disease in humans and animals . B . pertussis is the causative agent of whooping cough [8], which is responsiblefor up to 500,000 annual deaths worldwide in unvaccinated populations[52] and is increasing in incidence in some countries with establishedvaccination programs, including the United States [e.g., seereferences 15, 30, and 38] . Although most Bordetella speciescan cause similar disease in the upper respiratory tract, theirhost specificities vary dramatically . B . pertussis infects onlyhumans; B . parapertussis strains can be classified in two groups,one of which infects only humans and one of which infects onlysheep [16, 17, 25]; B . bronchiseptica causes respiratory infections in a wide variety of mammals and birds but only rarely in humans [22, 41, 51].

These three Bordetella species are closely related at the nucleotide sequence level [4, 24], and their relatedness has been furtherestablished using a variety of common molecular strain typingtechniques [reviewed in reference 29] . Phylogenetic analysesof the genus Bordetella, using pulsed-field gel electrophoresis,multilocus enzyme electrophoresis [MLEE], and IS typing data,suggested that B . pertussis, human-derived B . parapertussis,and sheep-derived B . parapertussis arose independently fromB . bronchiseptica-like ancestors [46, 47] . Comparative genomesequencing of a representative strain of each species [33] confirmedthat nucleotide sequence similarity is very high in conservedregions of the genome but demonstrated that B . pertussis andB . parapertussis evolved by genome decay from a B . bronchiseptica-like ancestor.

Although a number of critical conserved virulence mechanismshave been identified in the bordetellae, the genetic basis ofthese species' phenotypic variability remains unclear . We reasonedthat a comparative analysis based on genome content of numerousisolates of each species might clarify evolutionary relationshipsbetween the species and identify genes that determine key differencesin phenotypes, such as virulence and host specificity . Recentadvances in comparative genomic methods have illuminated theremarkable extent of variation between microbial genomes, evenamong strains of the same bacterial species [reviewed in references9, 20, 21, and 42] . We describe here the use of microarray-basedcomparative genome hybridization [CGH] and suppressive subtractivehybridization [SSH] to examine the relationships between genomecontent, host species, and pathogenicity.

 
Bordetella DNA microarray design and construction. Three thousand six hundred ninety-one probes representing 93%of the B . pertussis Tohama I predicted coding sequences wereamplified by PCR . More than 95% of the B . pertussis-derivedprobes were at least 95% identical to a sequence in the othertwo genomes . An additional 589 probes were amplified for B.bronchiseptica RB50 and B . parapertussis 12822 predicted codingsequences that were not at least 95% similar to an existingprobe . Microarrays were constructed using standard techniques,printing each probe at least twice on every array . Completedetails are in supplementary methods . [All supplementary materialsreferred to within this article are available at http://relman.stanford.edu/supplements/CGH2003.]

Strains and genomic DNA preparation. Bordetella strains used in this study are described in supplementaryTable 1 [see the above URL] . Single colonies of purity-confirmedcultures were inoculated into modified Stainer-Scholte mediaand grown to saturation prior to harvesting . Genomic DNA preparationis described in supplementary Methods [see the above URL].

Fluorescence labeling of genomic DNA. Genomic DNA was labeled by incorporation of dye-coupled nucleotidesbased on protocols found at the Brown Lab Mguide website [http://cmgm.stanford.edu/pbrown/protocols/index.html]. Test DNA samples were labeled with Cy5 . The mixed reference sample, comprising a 1:1:1 mixture of genomic DNA from B . pertussis Tohama I, B . parapertussis 12822, and B . bronchiseptica RB50,was labeled with Cy3 . Two micrograms of genomic DNA [in 10 mM Tris] was combined with 20 µl of 2.5x random octamers[BioPrime labeling kit; Invitrogen, Carlsbad, Calif.] in a totalvolume of 41 µl . The DNA was boiled for 5 min, placed on ice for 2 min, and centrifuged at 16,000 x g . The DNA wasmixed with 5 µl of deoxynucleoside triphosphate [dNTP]mix [120 nM [each] dATP, dCTP, and dGTP; 60 nM dTTP in Tris-EDTA],2.5 µl of 1 mM Cy5-dUTP or Cy3-dUTP [Amersham Biosciences,Little Chalfont, Buckinghamshire, England], and 1 µl of Klenow [40 U/µl; BioPrime labeling kit; Invitrogen] . The reaction was incubated at 37°C for 2 h and then stoppedby addition of 5 µl of 0.5 M EDTA [pH 8.0].

Microarray hybridization and scanning. Hybridization was performed as described in protocols foundat the Brown Lab Mguide website [http://cmgm.stanford.edu/pbrown/protocols/index.html]. Test and reference labeled DNA samples were combined and then washed two times using Microcon YM-30 columns [Millipore, Billerica, Mass.], according to the manufacturer's instructions . One hundred fifty micrograms of yeast tRNA [ATCC, Manassas, Va.] was addedto block nonspecific binding . The probe volume was adjustedto 12 µl with water, and then 2.55 µl of 20x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.45µl of 10% sodium dodecyl sulfate were added . Fourteenmicroliters of the probe was placed on the array and covered with a 25- by 25-mm no . 1 glass coverslip . The arrays were sealed in hybridization chambers [Die-Tech, San Jose, Calif.] with20 µl of 3x SSC to maintain humidity and incubated at65°C for 2 h to overnight.

Arrays were washed in 0.5x SSC, 0.005% sodium dodecyl sulfatefor approximately 30 s, 0.1x SSC for 30 s, 0.05x SSC for 1 min, and 0.025x SSC for 1 min . The first wash was performed at 65°C,and the remaining washes were performed at room temperature.Slides were dried by centrifugation for 5 min at 50 x g andscanned using a GenePix 4000B scanner and GenePix 3.0 software[Axon Instruments, Union City, Calif.].

Data filtering. Spots were screened visually to identify those of low quality.Signals from the remaining spots were converted into Cy5/Cy3ratios after subtracting local background intensity from eachspot in both channels . The ratios for each experiment were normalizedby adding a constant to the Cy5 signal so that the mean ratioacross the array was 1.0 . The data set was filtered to include only spot measurements with a net Cy3 [reference DNA] fluorescence intensity above 50 U, greater than 60% of pixels with intensity greater than one standard deviation above the mean Cy3 background intensity, and with usable data from at least 26 of the 42 arrays. Background-subtracted hybridization signals from replicate probes were averaged . After image analysis and data filtering, 4,140 independent probes remained in the data set.

Normalization against mixed reference. The mixed reference DNA sample contained equal amounts of genomicDNA from each of the three sequenced strains, so each probesequence was represented in proportion to its presence in oneor more of the sequenced strains . The reference signal was normalizedto account for differences in predicted gene abundance in thereference mixture . Based on hybridization experiments, a probesequence was considered absent from a sequenced strain if itstop BLASTN hit had less than 80% sequence identity over at least100 bp . For each BLAST hit exceeding 80% identity the speciessequence dose, d, was calculated as d = [% identity - 80]/20.The total sequence dose, D, in the reference DNA sample wascalculated as the sum of d from all three genomes, divided by3 . For each probe, the background-subtracted Cy5/Cy3 intensityratio was multiplied by its D value to obtain measurements normalizedfor sequence copy number in the reference.

Determination of threshold log intensity ratios. Because data distributions varied from experiment to experiment,the threshold was calculated independently for each individualarray based on its data distribution . Let x = 0.0025n where n is the number of data points from a single experiment . In this data set, x ranged from 8.94 to 10.33 . For each array,a histogram of the log ratio values with a bin size of 0.1 was computed . The bin value of the first bin to the left of the"main" peak [centered near zero] that had fewer than x measurements was chosen as the threshold . In other words, the threshold was selected by determining the point at which the left tail ofthe distribution representing the "detected" sequences containedvery few data points . The decision to use 0.0025 as the multiplierfor the calculation of x was empirically determined to maximizethe sensitivity and specificity for arrays of the three sequencedstrains [see Results].

For determination of sequences that vary between or within species, a conservative approach was used to further refine "not detected" calls . Reasoning that rare false-positive calls were unlikely to cluster by species or genome order, "undetected" data pointswere counted only if the same probe was not detected in a secondstrain of the same species or an adjacent probe sequence wasnot detected in the same strain.

Phylogenetic analysis. The data set was recoded as a binary representation of detected[1] and undetected [0] genes based on whether the log ratiohybridization signal was greater than or less than the thresholdratio [see above], respectively . Maximum-parsimony analysisof the data set was performed using PAUP [version 3.0; SinauerAssociates] with a Camin-Sokal model [13] . This algorithm waschosen because it requires no assumption that evolutionary ratesare equivalent among lineages . All character states were assumedto evolve independently, although a more exacting model wouldtreat the absence of adjacent genes as a single deletion event.The complete gene complement was designated the ancestral state[state = 1], and the undetected genes resulting from deletion events were a derived state [state = 0], which produced a rooted phylogenetic tree . Bootstrap values [18] were calculated from100 bootstrapped datasets using the BOOT, MIX, and CONSENSEprograms of the PHYLIP software package [version 3.5c [http://evolution.genetics.washington.edu/phylip/]].

Phylogenetic analysis treated each identified prophage as asingle character state . This was done because inclusion of multiple sequences from each prophage would give unwarranted weight to prophage loss events and potentially bias the phylogenetic analysis. Furthermore, bacteriophage are inherently mobile and possibly exchanged among Bordetella strains by lateral transfer and therefore do not accurately represent the evolutionary history of the core genome.

Confirmation of microarray-identified regions of difference. Ten regions of difference [RDs] were chosen randomly from regions where at least two adjacent sequences were variable betweenthe sequenced strain and another strain of the same speciesand thus were not detected using genome sequence data . Fourof these regions were variable among B . pertussis strains, threewere variable among B . parapertussis strains, and three werevariable among B . bronchiseptica strains . Primers were designedflanking one junction of 10 putative RDs . Primer sequences arein supplementary Table 2 [see the above URL] . For each RD, PCRwas performed with one strain for which the region was expectedto be present and one strain for which it was expected to beabsent . In addition, a conserved fragment of approximately 3kb was amplified from each DNA preparation to confirm that DNAwas amplifiable . Each PCR contained 1x GC genomic PCR buffer,1 M GC Melt, 1.1 mM Mg[OAc]₂, 0.2 mM dNTPs, and 1x Advantage-GC genomic polymerase mix [all from BD Biosciences Clontech, PaloAlto, Calif.], plus 200 nM [each] primer and 1 ng of genomicDNA, in a reaction volume of 25 µl . Touchdown PCR cyclingconditions were as follows: initial denaturation at 95°Cfor 1 min; 10 cycles of 94°C for 15 s, 65°C [decreasedby 1°C per cycle] for 30 s, 68° for 3 min; 25 cyclesof 94°C for 15 s, 55°C for 30 s, 68° for 3 min; and a final extension at 68°C for 3 min . PCR products wererun on agarose gels and visualized with ethidium bromide toconfirm the presence of a band of the expected size.

In every case tested, no PCR product of the expected size was detected in a strain predicted to be missing an RD . In one instancea larger PCR product was detected, suggesting that the strainmissing the RD might possess an insertion that occurred closeto the junction and disrupted or deleted the sequence foundat that position in the sequenced strain.

Subtractive hybridization. Subtractive hybridization was carried out with Bpe159 as thetester and a mixed driver pool of genomic DNA from the threesequenced strains of Bordetella . The PCR-Select bacterial genomesubtraction kit [Clontech] was used, with the following modificationsto optimize the protocol for the high-GC genome of Bordetella:the incubation temperatures of the two rounds of hybridizationwere increased to 66°C, 1.3% dimethyl sulfoxide and 1.3M betaine were added to the primary PCR, the primary PCR wasdiluted 1:15 instead of 1:40 for use as the secondary PCR template,and 12 to 15 cycles of amplification were performed for thesecondary PCR . After the generation of a clone library containing the products of the subtractive hybridization, inserts were screened for presence in Bpe159 . This was accomplished by amplifying approximately 300 inserts and printing them on microarrays.These screening microarrays were hybridized with Bpe159 andmixed reference genomic DNA . No spots had ratios significantlyhigher than the average . The two spots with the highest ratioof Bpe159/mixed reference signal were sequenced to confirm thatthey were present in both DNA pools.

In addition, B . pertussis strain 18323 was subjected to SSH analysis against B . pertussis Tohama I using the same methodology. Approximately 40% of the 300 fragments printed on the screening microarrays had a high ratio of 18323/Tohama I signal and werechosen for sequencing.

Amplification of fim2. Oligonucleotides were designed to amplify by PCR either theTohama I or RB50 fim2 gene [supplementary Table 2] . The forwardprimer was in a conserved region at the 5' end, and two reverseprimers at the 3' end were specific for the two genes [supplementaryTable 2] . The PCR mix contained 1x PCR buffer II [Applied Biosystems,Foster City Calif.], 1.5 mM MgCl₂, 0.6 mM dNTPs, 20 pmol ofeach primer, 1 ng of genomic DNA, and 1.25 U of AmpliTaq DNApolymerase [other than buffer, PCR reagents were from Perkin-Elmer,Boston, Mass.] . The reaction conditions were 96°C for 3 min; 35 cycles of 95°C for 1 min, 57°C for 1 min, 72°Cfor 1 min; and a final extension at 72°C for 3 min . PCRproducts were run on 2.5% agarose gels and visualized with ethidiumbromide staining.

Nucleotide sequence accession numbers. The sequences of two fim2 amplification products have been submittedto GenBank under the accession numbers AY289620 [Bpp4] and AY289621[Bbr77].

 
Interspecific comparative genome hybridization. A PCR product-based DNA microarray representing 93% of B . pertussis Tohama I, 75% of B . parapertussis 12822, and 73% of B . bronchiseptica RB50 predicted coding sequences was designed using draft genome sequence data [see the above URL for supplementary methods].Using this array, a collection of 42 B . pertussis, B . parapertussis, and B . bronchiseptica strains [supplementary Table 1] was surveyed by CGH . The 10 B . pertussis strains included 7 low-passage-number clinical isolates collected between 1989 and 1997 in the United States and The Netherlands and the laboratory strains TohamaI, Wellcome 28, and 18323 . The 10 B . parapertussis strains comprised 3 strains isolated from sheep and 7 strains isolated from humans. The 22 B . bronchiseptica strains were isolated from a variety of host species, including one human . The labeled genomic DNA of each strain was hybridized to the microarray together witha reference composed of a 1:1:1 mixture of genomic DNA fromeach of the three sequenced species . Comparison of the BordetellaCGH profiles revealed extensive variation between and withinspecies [Fig . 1 and supplementary data] . Bordetella strains exhibited multiple RDs, defined as blocks of two or more adjacent probes that hybridize poorly to at least one strain [Fig . 1].

 
For phylogenetic analysis and enumeration of variable genes,a threshold log ratio was calculated for each hybridizationexperiment and used to differentiate detected sequences fromundetected sequences, such that probes with values below thethreshold were called undetected . Accuracy of the method wasestimated by comparing, for the three sequenced genomes, callsbased on hybridization data to the "gold standard" of sequencedata [33] . A sequence was classified as "known absent" basedon sequence data if it had less than 80% similarity to the microarrayprobe sequence . For all three sequenced genomes, sensitivity[sequences correctly called "not detected" from array data/totalknown absent sequences] and specificity [sequences correctlycalled "detected"/total known present sequences] were calculated.Detection sensitivity for the three sequenced genomes rangedfrom 94.5 to 100%, and specificity ranged from 98.8 to 99.1%[supplementary Table 3 [see the above URL]] . Additionally, twodupilcate hybridizations of a B . pertussis genome were comparedto determine reproducibility of the method . Overall, 99% ofthe gene calls were concordant between the replicates.

Phylogenetic analysis of Bordetella. Variation in patterns of gene content often provides importantinsights into microbial evolution and population structure [21]. Maximum parsimony, previously used to analyze CGH datasets [36, 40], was employed for phylogenetic analysis of our CGH data.Because gene acquisition appears to be infrequent in Bordetella[33, 49], polarity was assumed such that the complete gene complementcomprised the ancestral state and deletion events were an irreversiblederived state . This analysis produced two equally likely phylogenies[Fig. 2, large tree and inset] . Both support the proposition that B . pertussis and B . parapertussis are more closely relatedto separate lineages of B . bronchiseptica than to each other[47] and that human and sheep isolates of B . parapertussis derivefrom a common ancestor but form separate clades . Although B.pertussis is monophyletic, the 18323 strain is very distantlyrelated to the others, as previously suggested by other typingmethods [10, 43, 47]; 18323 may represent a B . pertussis subspecies.IS element distribution is also useful for determining evolutionaryrelationships . IS1663 was detected in all B . pertussis strainsand in two B . bronchiseptica isolates [Fig . 2] . IS1001 was previously detected in B . parapertussis and a subset of B . bronchiseptica strains . The presence of IS1002 in B . pertussis and human-derivedB . parapertussis has been proposed to result from lateral transferof IS1002 to B . parapertussis from a coinfecting B . pertussis[46] . The simplest explanation for IS distribution is that afteran early split in the B . bronchiseptica lineage, one group acquiredIS1663 and then eventually gave rise to B . pertussis; the othergroup acquired IS1001 [which was sporadically lost from B . bronchiseptica] and then eventually gave rise to B . parapertussis . This is consistentwith the topology of the large tree in Fig . 2, which we assertto be the more likely . In this phylogeny, B . pertussis is mostclosely associated with a distinct clade of B . bronchisepticathat conspicuously includes the only human isolate in the study.The CGH-based trees are also consistent with 16S ribosomal DNA-basedphylogenies.

 
Our CGH-based phylogeny shares similarity with a MLEE-basedtree that supported the independent derivation of B . pertussis, human-derived B . parapertussis, and sheep-derived B . parapertussis from the B . bronchiseptica lineage [47]; however, the CGH-basedtree strongly supports the conclusion that the two subspeciesof B . parapertussis are more closely related to each other thanto any B . bronchiseptica strain . The CGH-based phylogeny, supportedby IS typing data, also identifies a deep-branching clade ofB . bronchiseptica that is associated with B . pertussis, whichis not supported by the MLEE-based tree; in fact, the threemembers of this clade represent two electrophoretic types [ET14and ET29] that differ at 6 out of 15 loci . These discrepanciesmay be due to differences in phylogenetic methods but may alsoreflect the relatively small number of gene content differencesbetween individual B . bronchiseptica strains.

Gene content diversity. To understand the molecular differences that distinguish theBordetella species, the subset of genes that was not detectedin at least one strain of each species was determined [see supplementarymethods] . Based on our phylogenetic analysis, human- and sheep-derivedB . parapertussis were treated separately . Eight hundred twentyprobes were not detected in at least one B . pertussis strain,and 93.9% of these were located in RDs; 477 [88.7% in RDs] and440 [86.4% in RDs] were not detected in at least one human-derivedand sheep-derived B . parapertussis strain, respectively; and555 [87.4% in RDs] were not detected in at least one B . bronchisepticastrain . Although RDs were sometimes located at similar RB50coordinates in multiple species, the endpoints of the RDs wereoften slightly different between species, suggesting that similardeletions arose independently in the different lineages . Atleast seven RDs represent either intact or cryptic prophage.Ten randomly chosen RDs were independently confirmed by PCRanalysis [supplementary methods and supplementary Table 2].Functional categorization of the genes that were not detectedin each species indicated that most encode cell envelope, periplasmicand exported proteins, global regulatory factors, transportproteins, laterally acquired elements [prophage], and hypotheticalopen reading frames [ORFs] [supplementary Fig. 1] . A caveatfor interpretation of these data is that gene inactivation byframeshift, point mutation, IS insertion, or regulatory mutationis not discernible by hybridization, so CGH may underestimatethe number of nonfunctional genes . For example, although theptx-ptl locus, encoding pertussis toxin, is detected by CGHin all three species [data not shown], it is known to be transcriptionallysilent in B . parapertussis and B . bronchiseptica [5], presumablydue to a small number of single-nucleotide differences in theptxA promoter region [33].

Species-specific sequences. Evolution of pathogenesis often involves the acquisition ofa small number of genes that enhance virulence or influencehost range [32, 53] . Eleven ORFs and two intergenic regionsfrom B . pertussis Tohama I were unique to B . pertussis [supplementary Table 4] . Nine ORFs fell into two contiguous gene clusters. With 61 to 63% GC content, both of these clusters deviate fromthe genome average [67.7%], suggesting that they may have been horizontally acquired . Each region contains a racemase fragment interrupted by an IS481 element in Tohama I, suggesting that recombination may have rearranged an ancestral insertion containing all nine genes . These sequences were not detected in B . pertussis 18323, indicating that the putative acquisition event occurred after divergence of this strain . Because 18323-like strainshave been isolated from pertussis patients as recently as 1993[10], these genes may not be necessary for B . pertussis pathogenesis or human adaptation . Another B . pertussis-specific gene, BP0507, encodes a hypothetical protein that is 46% similar to an internal region of the P.69 fragment of B . pertussis pertactin, an important adhesin and protective antigen.

One hundred forty-three sequences represented on the array were uniquely detected in B . bronchiseptica [supplementary Table 4] . Sixty of these genes, mostly prophage components, were detected only in the sequenced RB50 strain . Among the 83 remaining B. bronchiseptica-specific sequences, 58 are prophage components. The remaining 25 probes represent 21 ORFs . These include BB0124to BB0127 and BB0130, located in a contiguous block just upstreamof the proposed lipopolysaccharide [LPS] O-antigen biosynthesislocus [see below], bbuR, which encodes a proposed transcriptional regulator of urease expression [28], and bfrA, which encodesa ferric siderophore receptor previously shown to be B . bronchisepticaspecific [6].

None of the probes on the array hybridized uniquely to either human- or sheep-derived B . parapertussis . Because only 75% of the B . parapertussis 12822 genome was represented on the array, B . parapertussis-specific genes cannot be ruled out, but sequence alignment with the RB50 and Tohama I genomes identified a maximum of 33 possible B . parapertussis 12822-specific ORFs in the unrepresentedfraction of the 12822 genome, some of which may be present inother B . bronchiseptica or B . pertussis strains . Furthermore,a sheep-derived B . parapertussis strain has not yet been sequencedand may contain genes not present in the three sequenced genomes.

Molecular discrimination of Bordetella species in the clinical setting is imperfect . The ideal diagnostic markers would be present in every strain of the target species and absent fromall strains of the other species . In this data set, two B . pertussis sequences and three B . bronchiseptica sequences met these criteria. The B . pertussis-specific sequences were BP3385 and the B . pertussisBP0426-BP0427 intergenic region . The B . bronchiseptica-specificsequences were bfrA, BB4708, and fimN, although only the 3'end of fimN is unique . These sequences could potentially beused as the basis for a sensitive and specific diagnostic assay.

Intraspecific variation. B . pertussis is relatively homogeneous by some typing techniques[e.g., MLEE [47]] but exhibits heterogeneity in pulsed-fieldgel electrophoresis patterns [7, 23], probably due to extensive genome rearrangements [44] . Among the B . pertussis strains examinedin this study, 414 sequences were variably detected . The mostdivergent B . pertussis strain in this study was 18323, consistentwith results with other typing methods [4, 10, 43, 47] . This strain apparently represents an independently evolving branch of the B . pertussis lineage with very low representation in human infections . While this variant is pathogenic in humans[10], it is important to appreciate its genomic uniqueness whenusing strain 18323 to study B . pertussis pathogenesis . When18323 was omitted from the analysis, only 181 sequences werevariable, and 124 of these clustered within 26 RDs . All of thesequences that were variably detected among B . pertussis strainswere also detected on the RB50 chromosome . In the Tohama I genome,many of these RDs are flanked by IS elements, suggesting thatthese deletions may have resulted from recombination betweenperfect repeats . The stability of gene content in B . pertussisis notable given the high frequency of genome rearrangementobserved in natural populations [44] . The majority of B . pertussis-variable genes with assigned functions encode cell envelope components, exported, periplasmic, and lipoproteins, transport proteins,carbon and fatty acid degradation enzymes, and transcriptionalregulators . Of potential relevance to pathogenesis, severaliron acquisition genes were variably detected . A locus encodingthe ferric citrate uptake regulators, FecI and FecR, and threeputative iron uptake proteins was absent from six B . pertussisstrains . Probes in the alcaligin biosynthesis operon showedconsistent but small decreases in hybridization in three B.pertussis strains, as well as other strains throughout the genus,suggesting that the nucleotide sequence might be divergent.

The most striking observation about the B . parapertussis strains was the clear distinction between human- and sheep-derived isolates. Several RDs distinguished these two subspecies, including ORFs encoding transcriptional regulators, cell surface proteins,and transporters . The genomic region from cheR/cheX to tar/cheM was not detected in sheep-derived B . parapertussis . If chromosomal order is conserved in all B . parapertussis strains, this apparent deletion removes several components of the chemotaxis and motility apparatus, which may explain why sheep-derived B . parapertussis strains are nonmotile [2].

Within each of the two B . parapertussis subspecies, gene content was highly conserved, with only 105 variably detected sequences in the human-derived strains and 71 variably detected sequencesin the sheep-derived strains . The variable genes with assignedfunctions encode cell envelope, exported proteins, transporters,and transcriptional regulators . In one sheep-derived B . parapertussis isolate, genes encoding components of the type III secretion apparatus and an adjacent sigma factor were not detected, suggesting that type III secretion may not be required for pathogenesisof B . parapertussis in sheep . Genes encoding two putative adhesins, fhaS and BP3204/BPP0104, were variably detected among the B. parapertussis strains, implying a diversity of adherence mechanisms within this species.

B . bronchiseptica, with 453 variable sequences, exhibited the most heterogeneity of gene content among the species examined. The genome of B . bronchiseptica RB50, which has been isolated from the environmental gene pool as a result of laboratory passage, harbors three putative prophage that are not found in any other strain in this survey and a fourth prophage that is fully intactonly in this strain [Fig . 1] . Aside from its unusual prophage content, RB50 appeared to be a typical B . bronchiseptica strain. The presence of two other prophage was also variable withinthe species . The other variable genes with assigned functionsprimarily encode cell envelope, exported proteins, transporters,and transcriptional regulators . The putative adhesin BP3204/BPP0104was not detected in three B . bronchiseptica isolates or sheep-derived B . parapertussis . Seven B . bronchiseptica strains exhibited slightly reduced hybridization across the alcaligin biosynthesis locus, as observed for B . pertussis.

Variation in surface molecules. LPS is the predominant cell-surface glycolipid of gram-negativebacteria and an important virulence factor for many pathogens.Gene content of the Bordetella LPS biosynthetic locus was highlyvariable among the strains [Fig . 3] . As previously observed[37], the O-antigen biosynthesis locus was not detected in anyB . pertussis strains . BB0124 to BB0127 and B0130 were not detected in 12 B . bronchiseptica isolates and all B . parapertussis isolates.The LPS O-antigen locus previously reported for B . bronchisepticastrain C7635E [37] differs in this region from the RB50 locus[33] . Because probes for the wbmPQRS genes identified in C7635Ewere not included on the array, the presence of these genescould not be determined, so alternative ORFs may be presentin this locus in some B . bronchiseptica strains . The ORFs inthis region may encode proteins, formyltransferases in particular,that modify the terminal sugar of O antigen [48] . Genomes oftwo B . bronchiseptica strains and all sheep-derived B . parapertussis strains did not hybridize to BB0131 to BB0136 [wbmIJKLMN] and BB0140 to BB0141 [wbmDE] . These strains may lack key O-antigen biosynthetic enzymes and transport proteins, which could result in variant LPS structures . Three other B . bronchiseptica strains had unique LPS locus gene content patterns . These results provide a molecular explanation for LPS structural diversity between and within Bordetella species [45] and could have importantramifications for virulence and host association.

 
Bordetella autotransporters [e.g., BrkA, Vag-8, and SphB1] also have been implicated in host cell interactions and virulence [reviewed in reference 26] . This family of proteins is distinguishedby the presence of a shared domain that inserts into the gram-negativebacterial outer membrane and translocates the attached domainto the cell surface . Several of the Bordetella autotransporterswere variably detected [supplementary Fig. 2], including bapA,bapC, and three uncharacterized proteins.

Fimbriae are Bordetella adhesins, major antigens, and determinants of serotype [50] . Absence of both fimN and a novel putativefimbrial subunit, BB3424/BPP1684, was confirmed by CGH for allB . pertussis strains [Fig . 4] . Sequence divergence as low as2% could be discerned visually as blocks of consistent, slightlynegative log ratios within the species that diverged from theprobe sequence . fim2 was represented by two probes that areonly 67% identical . The RB50 fim2 probe hybridized to all B.bronchiseptica and B . parapertussis strains but not to B . pertussisstrains . Unexpectedly, the Tohama I-derived fim2 probe hybridizedto all three ovine B . parapertussis strains and 16 of the 22B . bronchiseptica strains, as well as to the B . pertussis strains.This indicated that sheep-derived B . parapertussis strains andsome B . bronchiseptica strains might have both a Tohama I-like fim2 gene and an RB50-like fim2 gene . Species-specific fim2 PCR was performed on three sheep-derived B . parapertussis strains and four B . bronchiseptica strains that hybridized to both probes [supplementary methods] . Tohama I-specific primers failed to amplify a fragment, but PCR with the RB50-specific primers amplified a product in all seven strains [data not shown] . Amplicons froma sheep-derived B . parapertussis [Bpp4] strain and a B . bronchiseptica [Bbr77] strain were cloned and sequenced . The 518-bp Bpp4 gene fragment was 98% identical to the RB50 sequence and was predictedto encode a protein with a ThrArg substitution and a three-amino-aciddeletion relative to the RB50 protein [supplementary Fig . 3].The 528-bp Bbr77 fragment was 97% identical to the RB50 sequenceand encoded a protein with five amino acid substitutions relativeto the RB50 protein . Because these sequences are less than 63%identical to that of the Tohama I-derived probe, they do notaccount for the hybridization signals obtained with that probein the CGH experiments . Therefore, these strains probably possessa second fim2 gene that is similar to the Tohama I orthologue.Loss of fimbrial structural alleles in bordetellae may be responsiblein part for a restriction in host range.

 
Subtractive hybridization of B . pertussis strains. An important limitation of microarray-based CGH studies is thatonly sequences on the array can be assayed . To investigate thepotential genetic diversity in B . pertussis strains that havenot been sequenced, SSH experiments were performed . SSH of B.pertussis 18323, which is genetically distinct from Tohama I[31], against a Tohama I driver pool produced 91 clones thatdid not hybridize to Tohama I genomic DNA, all of which werehighly similar to sequences in the B . bronchiseptica RB50 orB . parapertussis 12822 genome [data not shown] . All of the sequences identified by SSH that were also represented as probes on the microarray were detected in 18323 and not detected in TohamaI, further verifying the accuracy of the methods . SSH of a recent clinical isolate, Bpe159, against a mixed driver pool comprising Tohama I, RB50, and 12822 produced 174 clones, all of whichupon screening hybridized to the driver pool . These resultsprovide further evidence that gene acquisition by B . pertussishas been rare.

Pathogen evolution by gene loss. Gene loss events may have driven the speciation of B . pertussisand B . parapertussis by altering regulatory networks, modifyingmetabolic pathways [27], or eliminating antigenic proteins thatmade them susceptible to immune surveillance . These genomicmodifications could have resulted in novel virulence characteristicsthat made the strains more effective at infecting a particularhost [e.g., humans] . Alternatively, genome decay may have occurredsubsequent to host restriction due to a loss of selective pressureagainst gene loss and deleterious transposition events [3, 32]. In this scenario, the initial niche restriction may have been attributable to a more subtle genetic change, such as inactivationor modification of a key surface molecule or regulatory factor.In fact, CGH revealed extensive variation in the complementof regulatory proteins and in the presence and sequence of genesresponsible for cell surface structures including LPS, fimbriae,and autotransporters . These two hypotheses are not mutuallyexclusive.

The decayed genomes of B . pertussis and B . parapertussis are also characterized by high IS element loads [46, 47] . Of particularnote, many inferred genomic rearrangements between B . bronchisepticaand the derived species are closely associated with IS elements[33], suggesting that recombination between identical copiesof IS elements is an important mechanism in the genesis of inversions, transpositions, and deletions . Genome rearrangement may be the inevitable consequence of accumulating high copy numbers ofone or more repetitive sequences . Adoption of a host-restrictedlifestyle may have relaxed selection against some deleterioustransposition events and subsequent rearrangements . Furthermore,modelling experiments have suggested that selection againstincreased transposon copy number could be diminished in smallpopulations, as might have been encountered by the originalB . pertussis and B . parapertussis progenitors [11].

Genome decay is frequently observed in the genomes of obligate intracellular pathogenic bacteria [3, 32], but among extracellularpathogens the inferred evolution of B . pertussis and B . parapertussisfrom a B . bronchiseptica-like ancestor is most similar to theproposed evolution of Yersinia pestis from Yersinia pseudotuberculosis[1, 34] . In both cases, relative to the ancestral species, the derived species exhibit extensive genomic deletions and rearrangements, accumulation of pseudogenes, and high copy numbers of IS elements that result in genomic fluidity . Genes encoding uptake and transport functions are often inactivated, while central and intermediate metabolism genes remain largely intact . The derived strainsalso share phenotypic features, including expression of roughLPS [37, 39, 45] and limited capability to survive outside thehost [1, 14, 35] . A fundamental difference is that gene acquisition has played a fundamental role in the evolution of Y . pestis [12] but does not appear to have contributed significantly toBordetella evolution . Instead, the molecular events that initiallyresulted in modified host adaptation and virulence traits inB . pertussis and B . parapertussis were probably modificationsor deletions of existing genes or genome rearrangements withglobal regulatory consequences.

By elucidating the molecular differences between the Bordetella species, we may achieve an understanding of how host adaptation occurs . Ultimately, our results could help to explain the mechanisms of vaccine-induced immunity, which may have played a role inthe reemergence of B . pertussis [30] and potentially to guidethe design of improved vaccines.

 
This work was funded by NIH grants R01 AI039587 and R03 AI547970to D.A.R . Microarray design software development was fundedby a Stanford University Office of Technology and LicensingInnovation Research Incentive Award . C.A.C . received ResearchTraining Fellowships from the American Lung Association [ALA]and ALA-California . M.M.B . was supported by a Smith StanfordGraduate Fellowship.

We are grateful to Julian Parkhill and the PSU of the Sanger Institute for making genome sequence assemblies and annotation available prior to publication . We also thank Frits Mooi, Gary Sanden, James Musser, Sylvia Yeh, and Jeff F . Miller for providing bacterial strains, Trevor Hastie, Hester Bootsma, and Dimitri Diavatopoulos for helpful discussions, Ikro Yoon for software development, Brett Petersen for protocol development, and Hassya Kedem, Jennifer Maynard, Christine Dieterich, Elisabeth Bik,and Isabelle da Piedade for assistance with array production.

 
* Corresponding author . Mailing address: VA Palo Alto Health Care System, 3801 Miranda Ave., 154T, Palo Alto, CA 94304 . Phone: [650] 852-3308 . Fax: [650] 852-3291 . E-mail: relman@cmgm.stanford.edu .

M.M.B . and C.A.C . contributed equally to this study.

Present address: Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.

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