pasteurellaceae genera actinobacillus, haemophilus ... · researcharticle phylogenomic and...

Research ArticlePhylogenomic and Molecular Demarcation ofthe Core Members of the Polyphyletic Pasteurellaceae GeneraActinobacillus Haemophilus and Pasteurella

Sohail Naushad Mobolaji Adeolu Nisha Goel Bijendra KhadkaAqeel Al-Dahwi and Radhey S Gupta

Department of Biochemistry and Biomedical Sciences McMaster University Hamilton ON Canada L8N 3Z5

Correspondence should be addressed to Radhey S Gupta guptamcmasterca

Received 5 November 2014 Revised 19 January 2015 Accepted 26 January 2015

Academic Editor John Parkinson

Copyright copy 2015 Sohail Naushad et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The genera Actinobacillus Haemophilus and Pasteurella exhibit extensive polyphyletic branching in phylogenetic trees and do notrepresent coherent clusters of species In this study we have utilized molecular signatures identified through comparative genomicanalyses in conjunction with genome based and multilocus sequence based phylogenetic analyses to clarify the phylogenetic andtaxonomic boundary of these generaWe have identified large clusters ofActinobacillus Haemophilus and Pasteurella species whichrepresent the ldquosensu strictordquo members of these genera We have identified 3 7 and 6 conserved signature indels (CSIs) which arespecifically shared by sensu stricto members of Actinobacillus Haemophilus and Pasteurella respectively We have also identifiedtwo different sets of CSIs that are unique characteristics of the pathogen containing genera Aggregatibacter and Mannheimiarespectively It is now possible to demarcate the genera Actinobacillus sensu stricto Haemophilus sensu stricto and Pasteurella sensustricto on the basis of discrete molecular signatures The other members of the genera Actinobacillus Haemophilus and Pasteurellathat do not fall within the ldquosensu strictordquo clades and do not contain thesemolecular signatures should be reclassified as other generaThe CSIs identified here also provide useful diagnostic targets for the identification of current and novel members of the indicatedgenera

1 Introduction

The familyPasteurellaceae the single constituent family of theorder Pasteurellales represents a diverse group of commensaland pathogenic bacteria within the class Gammaproteobacte-ria The family currently contains 19 genera some of whichare particularly important human and animal pathogens [12] The genera Haemophilus contains species responsible forhuman bacteremia pneumonia acute bacterial meningitisand the sexually transmitted disease chancroid [3ndash5] Aggre-gatibacter species have been implicated in juvenile periodon-titis [6] members of the genera Mannheimia Pasteurellaand Actinobacillus have been implicated in the causation ofshipping fever in cattle fowl cholera and pleuropneumoniain pigs respectively [7ndash9]

The family Pasteurellaceae was originally proposed as ahigher level taxonomic grouping of the related pathogenicgenera Actinobacillus Haemophilus and Pasteurella [10]

Classification of organisms into these three genera wasprimarily based on DNA G-C content and a handful ofphenotypic traits [11] The phenotypic traits were later foundnot to be characteristic of any single genus [12] Consequentlythe genera Actinobacillus Haemophilus and Pasteurella eachexhibit extensive polyphyly in subsequent 16S rRNA basedphylogenies [12 13] Additional studies based on individ-ual or concatenated gene sets and DNA-DNArRNA-DNAhybridization also support the presence of extensive poly-phyly within the genera Actinobacillus Haemophilus andPasteurella [14ndash20]

Extensive work has been undertaken to amend theclassification of the genera Actinobacillus Haemophilus andPasteurella [1 5 9 18] New genera have been created tohouse phylogenetically coherent clusters of ActinobacillusHaemophilus and Pasteurella The species [Actinobacillus]actinomycetemcomitans [Haemophilus] aphrophilus [Hae-mophilus] paraphrophilus and [Haemophilus] segnis have

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2015 Article ID 198560 15 pageshttpdxdoiorg1011552015198560

2 International Journal of Genomics

been transferred to the genus Aggregatibacter [21] thespecies [Haemophilus] paragallinarum [Pasteurella] galli-narum [Pasteurella] avium and [Pasteurella] volantium havebeen transferred to the genus Avibacterium [22] the species[Haemophilus] somnus and [Haemophilus] agni have beentransferred to the genus Histophilus [23] and the species[Pasteurella] haemolytica and [Pasteurella] granulomatis havebeen transferred to the genus Mannheimia [7] Additionallysome individual species within the genera ActinobacillusHaemophilus and Pasteurella that do not cluster with othermembers of their genus in phylogenetic trees have beenmoved or proposed to be moved to novel or neighbouringgenera (namely the transfer of the species [Haemophilus]pleuropneumoniae to the genus Actinobacillus [24] the trans-fer of the species [Pasteurella] anatis to the genus Gallibac-terium [25] the transfer of the species [Pasteurella] trehalosito the genus Bibersteinia [26] the transfer of the species [Pas-teurella] ureae to the genus Actinobacillus [27] and the pro-posed transfer of the species [Haemophilus] ducreyi to a novelgenus [28]) However despite these changes the classificationof the genera Actinobacillus Haemophilus and Pasteurellais still problematic and each genus continues to containmembers which exhibit polyphyletic branching [5 17ndash20]

Multiple studies have attempted to define a core group ofspecies which cluster around the nomenclatural type speciesofActinobacillus Haemophilus or Pasteurella as the only truemembers of these genera (ie sensu stricto) [13 15ndash17 29ndash31]but the taxonomy and phylogeny of these bacteria continue toremain inconclusive [20 32 33] Several methods have beenemployed for the demarcation of these genera however nosimplemethod or criterion is available that can clearly delimitthese genera It has been suggested that genome based studiesmay provide reliable means of clarifying the evolutionaryrelationships of these bacteria [33]

Since the availability of the first complete genomesequence of theHaemophilus influenzae [34] a large numberof genomes for the members of the family Pasteurellaceaehave become available in public databases [35 36] The avail-ability of these genomes provides us with an opportunity tocomplete comprehensive genome scale phylogenetic analysesof the family Pasteurellaceae These genome sequences havealso been utilized to carry out comparative genomic analysesto identifymolecular signatures (namely conserved signatureindels (CSIs) in various proteins) commonly shared by allor closely related subsets of species within the family Pas-teurellaceae On the basis of the molecular signatures iden-tified from comparative analyses of Pasteurellaceae genomesin conjunction with core genome based and multilocussequence based phylogenetic analyses we have identifiedsensu stricto clades of Actinobacillus Haemophilus and Pas-teurella that are supported by 3 7 and 6 unique molecularsignatures respectively We also report sets of molecularsignatures that are unique characteristics of the pathogencontaining genera Aggregatibacter andMannheimia

2 Methods

21 Multilocus Sequence Analysis Multilocus sequence anal-ysis was completed for members of the family Pasteurellaceae

using widely available nucleotide sequences of the 16S rDNAinfB (translation initiation factor IF-2) recN (DNA repairprotein) and rpoB (DNA-directed RNA polymerase subunitbeta) genes which have been used individually or as partof a set in a number of previous phylogenetic analyses ofthe family Pasteurellaceae [15ndash17 29 30] Gene sequences forthese four genes were obtained for 52 Pasteurellaceae strainsrepresenting a large majority of the known Pasteurellaceaespecies and 2 members of Vibrio cholerae from the NCBInucleotide database [37] Species which were missing one ofthese four genes or which did not have a gene sequence thatwas at least 50 of the length of the full gene were excludedfrom the analysis The four genes were individually alignedusing MUSCLE [38] and manually concatenated to create acombined dataset that contained 10 183 nucleotide long align-ments A maximum-likelihood tree based on 100 bootstrapreplicates of this alignment was constructed using MEGA60 [39] while employing maximum composite likelihoodsubstitution model

22 Pasteurellaceae Core Genome Phylogenetic Tree Aphylo-genetic tree of 76 Pasteurellaceae strains rooted using 7mem-bers of the family Vibrionaceae based on the core genomeof the family Pasteurellaceae was created for this study Thecore set of Pasteurellaceae proteins were identified usingthe UCLUST algorithm [55] to identify widely distributedprotein families with at least 30 sequence identity and50 sequence length Proteins families which were presentin less than 50 of the input genomes were excluded fromfurther analysis Potentially paralogous sequences (additionalproteins from the same organism in a single protein family)within the remaining protein families were also excludedfrom further analysis Each protein family was individuallyaligned using MAFFT 7 [56] Aligned amino acid positionswhich contained gaps in more than 50 of organisms wereexcluded from further analysis The remaining amino acidpositionswere concatenated to create a combined dataset thatcontained 128 080 amino acid long alignments An approx-imately maximum-likelihood tree based on this alignmentwas constructed using FastTree 2 [57] while employing theWhelan and Goldman substitution model [58]

23 Identification of Molecular Signatures (CSIs) for DifferentGenera of the Family Pasteurellaceae The detailed outlineof the process of identifying CSIs has been recently pub-lished [59] In brief Blastp searches were performed onall proteins from the genome of Haemophilus influenzaeF3047 [47] Ten to fifteen high scoring homologues thatwere present in Haemophilus other Pasteurellaceae andGammaproteobacteria species were retrieved and their mul-tiple sequence alignments were constructed using Clustal X183 [60] The alignments were visually inspected to identifyany conserved inserts or deletions (indels) that are restrictedto the particular clades of the family Pasteurellaceae whichare flanked on each side by at least 5-6 identicalconservedresidues in the neighbouring 30ndash40 amino acidsThe selectedsequences containing the indels and their flanking conservedregions were further evaluated by detailed Blastp searches

International Journal of Genomics 3

to determine species distribution and group specificity Theresults of these Blast searcheswere processed using Sig Createand Seq Style to construct signature files [59] Due to spaceconstraints the sequence alignment files presented herecontain sequence information for a limited number of specieswithin the orderPasteurellaceae and a representative selectionof outgroup speciesHowever in each case allmembers of theorder and outgroups exhibited similar sequence characteris-tics to the representatives

3 Results and Discussion

31 Phylogenetic Analysis of the Pasteurellaceae Elucidatingan accurate phylogeny of themembers of the familyPasteurel-laceae has been a long standing challenge in Pasteurellaceaeresearch [10ndash12 18 19] Early 16S rRNAbased studies revealedthat the established taxonomy of the family Pasteurellaceaewas not consistent with their genetically inferred phylogeny[12 14] This has led to a long series of taxonomic revisionswithin the family Pasteurellaceae a process which is still tak-ing place today [7 18 22 28] However it was subsequentlydiscovered that phylogenetic trees of Pasteurellaceae speciesbased on different genes did not completely agree with eachother [15 16 31] In particular phylogenetic trees based onthe 16S rRNA gene often considered the gold standard inbacterial taxonomy and phylogeny [61 62] disagreed withhighly robust multilocus sequence and concatenated proteinsequence based phylogenetic trees [9 17 19 20 53 63]

Phylogenetic trees based on concatenated sequences fora large number of unlinked and conserved loci are morereliable and robust than phylogenetic trees based on anysingle gene or protein [64 65] Due to a rapid increasein the availability of genomic sequence data we are nowable to complete genome scale phylogenetic analyses of thefamily Pasteurellaceae which cover a vast majority of thediversity within the family In this work we have produceda phylogenetic tree for 74 genome sequenced members of thefamily Pasteurellaceae based on 128 080 aligned amino acidpositions (Figure 1(a)) The branching patterns of the coregenome phylogenetic tree produced in this work largely agreewith a previous genome based phylogenetic tree producedfor a limited number of Pasteurellaceae species [19] and aconcatenated protein based phylogenetic tree of the familyPasteurellaceae produced by our lab in a previous study [20]Additionally we have also produced a multilocus sequencebased phylogenetic tree using the 16S rDNA infB recN andrpoB genes which are commonly used in the phylogeneticanalysis of the family Pasteurellaceae (Figure 1(b)) [15ndash1729 30] This tree also showed broadly similar branchingpatterns to pastmultilocus sequence based phylogenetic trees[17 18] and to our core genome based phylogenetic treeBoth our core genome based and multilocus sequence basedphylogenetic trees provide evidence for a division of thePasteurellaceae into at least two higher taxonomic groups(families) which are broadly similar to the two clades ofPasteurellales identified in our previous work [20] A similardivision of the family Pasteurellaceae into two or morelarge groups is seen in many other robust multilocus orconcatenated protein based phylogenetic trees [17 19 53 63]

however this division is not readily apparent in phylogeniesbased on the 16S rRNA gene [9 66]

A majority of the known genera within the familyPasteurellaceae form well-defined and coherent clusters inphylogenetic trees (Figure 1) [9 17 19 20 66] The generaActinobacillus Haemophilus and Pasteurella which weredescribed before the advent of genetic characterizationexhibit polyphyletic branching in all gene and protein basedphylogenetic trees including the core genome based andmultilocus sequence based phylogenetic trees created in thiswork (Figure 1) However there are large clusters of Acti-nobacillus Haemophilus and Pasteurella species identifiablein the phylogenetic trees which represent the core or ldquosensustrictordquo members of each genera The clusters of speciesthat represent Actinobacillus sensu stricto Haemophilus sensustricto and Pasteurella sensu stricto are indicated in Figure 1Members of each genera which fall outside of the sensu strictoclusters indicated in our phylogenetic trees by the presence ofsquare brackets around their genus name (eg [Pasteurella]pneumotropica) are only distantly related to the sensu strictomembers of their genus and will require reclassification inorder to make their taxonomy and phylogeny concordant

32 The Usefulness of Conserved Signature Indels as Phylo-genetic and Taxonomic Markers Whole genome sequencesare a rich resource for the discovery of molecular signatureswhich are unique to a group of organisms [67ndash69] One usefulclass of shared molecular signatures are conserved signa-ture indels (CSIs) which are insertionsdeletions uniquelypresent in protein sequences from a group of evolutionarilyrelated organisms [59 70 71] The unique shared presenceof multiple CSIs by a group of related species is mostparsimoniously explained by the occurrence of the geneticchanges that resulted in these CSIs in a common ancestorof the group followed by vertical transmission of these CSIsto various descendant species [59 71ndash73] Hence these CSIsrepresent molecular synapomorphies (markers of commonevolutionary decent) which can be used to identify anddemarcate specific bacterial groups in molecular terms andfor understanding their interrelationships independently ofphylogenetic trees [59 70ndash72] CSIs have recently beenused to propose important taxonomic changes for a num-ber of bacterial groups (namely Aquificae SpirochaetesThermotogae Xanthomonadales and Borrelia) at differenttaxonomic ranks [69 74ndash77] In the present work we havecompleted comprehensive comparative analysis of Pasteurel-laceae genomes (Table 1) in order to identify CSIs thatare primarily restricted to the different genera within thefamily Pasteurellaceae We have identified 3 7 and 6 uniquemolecular signatures which are shared by Actinobacillussensu stricto Haemophilus sensu stricto and Pasteurella sensustricto respectively Information regarding these CSIs andtheir evolutionary significances is discussed below

33 Molecular Signatures Specific for Actinobacillus sensustricto The genus Actinobacillus was originally defined asa group of growth factor independent host-associated rodswhich shared phenotypic or biochemical similarity with


Haemophilus influenzae 224-21Haemophilus influenzae 7P49H1Haemophilus influenzae 86-028NPHaemophilus influenzae 6P18H1Haemophilus influenzae PittHHHaemophilus influenzae 10810Haemophilus aegyptius ATCC 11116Haemophilus influenzae F3031Haemophilus influenzae RdAWHaemophilus influenzae 221-21Haemophilus influenzae PittAAHaemophilus influenzae KR494

Haemophilus haemolyticus M19501Haemophilus haemolyticus M19107Haemophilus haemolyticus HK386Haemophilus sporal taxon 851 str F0397[Pasteurella] pneumotropica DSM 21403

Haemophilus parainfluenzae HK2019Haemophilus parainfluenzae ATCC 33392

Haemophilus pittmaniae HK 85

Haemophilus sensustricto

Aggregatibacter actinomycetemcomitans D11S-1Aggregatibacter actinomycetemcomitans ANH9381Aggregatibacter actinomycetemcomitans I23CAggregatibacter actinomycetemcomitans RhAA1Aggregatibacter actinomycetemcomitans SC1083Aggregatibacter aphrophilus ATCC 33389Aggregatibacter aphrophilus F0387Aggregatibacter segnis ATCC 33393Aggregatibacter sp oral taxon 458 str W10330

Aggregatibacter

Avibacterium paragallinarum 72Avibacterium paragallinarum 221Histophilus somni 2336Histophilus somni 129PT

Pasteurella dagmatis ATCC 43325Pasteurella multocida 1500EPasteurella multocida P1059Pasteurella multocida 1500CPasteurella multocida 671 90Pasteurella multocida 3480

Pasteurella sensu stricto

[Actinobacillus] succinogenes 130ZBasfia succiniciproducens MBEL55E[Pasteurella] bettyae CCUG 2042[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis[Haemophilus] parasuis

2975584-15995SH0165gx033ZJ0906MN-H12939SW114D74

Bibersteinia trehalosi USDA-ARS-USMARC-190Bibersteinia trehalosi USDA-ARS-USMARC-188[Haemophilus] paraphrohaemolyticus[Haemophilus] paraphrohaemolyticus

HK411HK385

[Haemophilus] sp HK 2154[Haemophilus] parainfluenzae CCUG 13788

[Actinobacillus] minor[Actinobacillus] minor

NM305202

Actinobacillus ureae ATCC 25976Actinobacillus suis H91-0380Actinobacillus capsulatus DSM 19761Actinobacillus pleuropneumoniae 1096Actinobacillus pleuropneumoniae L20Actinobacillus pleuropneumoniae 56153Actinobacillus pleuropneumoniae S8

Actinobacillus sensustricto

[Haemophilus] ducreyi 35000HPMannheimia varigena USDA-ARS-USMARC-1388Mannheimia varigena USDA-ARS-USMARC-1296Mannheimia varigena USDA-ARS-USMARC-1312Mannheimia varigena USDA-ARS-USMARC-1261Mannheimia haemolytica D35Mannheimia haemolytica D153Mannheimia haemolytica PKL10

Mannheimia

Gallibacterium anatis 1265612Gallibacterium anatis F 149

005

(a)


Pasteurella sensustricto


Haemophilus influenzae Rd KW20Haemophilus influenzae R2866Haemophilus influenzae 10810Haemophilus aegyptius CCUG 25716Haemophilus haemolyticus M21639Haemophilus pittmaniae HK 85

Haemophilus parainfluenzae T3T1[Pasteurella] pneumotropica DSM 21403

[Actinobacillus] porcinus NM319Lonepinella koalarum ATCC 700131Avibacterium paragallinarum JF4211Avibacterium gallinarum NCTC 11188[Haemophilus] felis ATCC 49733Pasteurella dagmatis ATCC 43325Pasteurella multocida ATCC 43137Pasteurella multocida HN06Pasteurella multocida 36950Aggregatibacter actinomycetemcomitans D11S-1Aggregatibacter aphrophilus NJ8700 Aggregatibacter

[Actinobacillus] succinogenes 130ZNecropsobacter rosorum CCUG 28028

Basfia succiniciproducens JF4016Basfia succiniciproducens MBEL55E

[Pasteurella] bettyae CCUG 2042Bisgaardia hudsonensis M327992[Haemophilus] sputorum HK 2154

Volucribacter psittacicida CCUG 47536[Actinobacillus] indolicus 46KC2[Haemophilus] parasuis KL0318Nicoletella semolina JF2465Bibersteinia trehalosi USDA-ARS-USMARC-192

[Actinobacillus] muris NCTC 12432Otariodibacter oris Baika1Vespertiliibacter pulmonis E12708[Haemophilus] paraphrohaemolyticus HK385[Haemophilus] paraphrohaemolyticus HK411Histophilus somni 2336

[Actinobacillus] minor NM 305Actinobacillus lignieresii NCTC 4189Actinobacillus pleuropneumoniae L20Actinobacillus arthritidis CCUG 24862Actinobacillus capsulatus DSM 19761Actinobacillus suis ATCC 33415Actinobacillus ureae CCUG 2139Mannheimia haemolytica D171Mannheimia varigena USDA-ARS-USMARC-1261Mannheimia granulomatis DSM 19156

Mannheimia

[Haemophilus] ducreyi 3500HPGallibacterium anatis F 149

Gallibacterium melopsittaci F450Chelonobacter oris 1662

[Pasteurella] testudinis CCUG 19802

01

(b)

Figure 1 (a) A maximum-likelihood core genome phylogenetic tree of sequenced members of the family Pasteurellaceae (b) A maximum-likelihood phylogenetic tree based on concatenated nucleotide sequence alignments of the 16S rDNA infB recN and rpoB genes Both treesare rooted using members of the Vibrionaceae (not shown) Nodes with gt80 bootstrap support are indicated by diamond shaped symbolsat the node Clusters of species representing Actinobacillus sensu stricto Haemophilus sensu stricto Pasteurella sensu stricto AggregatibacterandMannheimia are indicated by brackets Members of the genera Actinobacillus Haemophilus and Pasteurella which do not fall into theirrespective ldquosensu strictordquo clades are indicated by the presence of square brackets around their generic name (ex [Pasteurella] pneumotropica)


Table 1 Genome characteristics of the sequenced Pasteurellaceae included in our analyses

Organism name BioProject Size (Mb) Proteins G-C () ReferencesActinobacillus pleuropneumoniae L20 CP000569 227 2013 413 [40]Actinobacillus pleuropneumoniae serovar 3 str JL03 CP000687 224 2036 412 [41]Actinobacillus pleuropneumoniae serovar7 str AP76 CP001091 235 2142 412 STHHb

Actinobacillus ureae ATCC 25976a AEVG0 230 2475 mdash BCMg

Actinobacillus minor 202a ACFT0 213 2050 393 McGill UniversityActinobacillus minor NM305a ACQL0 243 2411 393 McGill UniversityActinobacillus succinogenes 130Z CP000746 232 2079 449 Joint Genome InstituteAggregatibacter aphrophilus NJ8700 CP001607 231 2219 422 [19 42]Aggregatibacter actinomycetemcomitans D11S-1 CP001733 220 2280 443 [43]Aggregatibacter actinomycetemcomitans D7S-1 CP003496 231 2250 443 [44]Aggregatibacter segnis ATCC 33393a AEPS0 199 1956 mdash BCMg

Gallibacterium anatis UMN179 CP002667 269 2500 399 [45]Haemophilus aegyptius ATCC 11116a AFBC0 192 2020 mdash BCMg

Haemophilus ducreyi 35000HP AE017143 170 1717 382 Ohio State UniversityHaemophilus haemolyticusM21621a AFQQ0 209 1894 mdash [46]Haemophilus influenzae 10810 FQ312006 198 1903 381 WTSIh

Haemophilus influenzae F3031 FQ670178 199 1770 382 [47]Haemophilus influenzae F3047 FQ670204 201 1786 382 [47]Haemophilus influenzae 221-21a AAZD0 189 2224 380 [48]Haemophilus influenzae 3655 AAZF0 188 1929 380 [48]Haemophilus influenzae 6P18H1a ABWW0 191 1893 382 CGS ASRIe

Haemophilus influenzae 7P49H1a ABWV0 183 1752 379 CGS ASRIe

Haemophilus influenzae NT127a ACSL0 187 1809 380 BIGSPc

Haemophilus influenzae PittAAa AAZG0 188 1981 381 [48]Haemophilus influenzae PittIIa AAZI0 195 2028 380 [48]Haemophilus influenzae PittHHa AAZH0 184 1977 380 [48]Haemophilus influenzae R3021a AAZJ0 188 2307 379 [48]Haemophilus influenzae RdAWa ACSM0 180 1718 380 BIGSPc

Haemophilus influenzae 86-028NP CP000057 191 1792 382 [49]Haemophilus influenzae PittEE CP000671 181 1613 380 [48]Haemophilus influenzae PittGG CP000672 189 1661 380 [48]Haemophilus influenzae Rd KW20 L42023 183 1657 382 [34]Haemophilus influenzae R2846 CP002276 182 1636 380 UW-SBRId

Haemophilus influenzae R2866 CP002277 193 1795 381 UW-SBRId

Haemophilus parainfluenzae ATCC 33392 a AEWU0 211 2010 mdash BCMg

Haemophilus parainfluenzae T3T1 FQ312002 209 1975 396 WTSIh

Haemophilus parasuis 29755a ABKM0 222 2244 398 Iowa State UniversityHaemophilus parasuis SH0165 CP001321 227 2021 400 [50]Haemophilus pittmaniaeHK 85a AFUV0 218 2390 mdash J Craig Venter InstituteHaemophilus sputorum CCUG13788a AFNK0 214 2073 mdash Aarhus University HospitalHaemophilus parahaemolyticusHK385a AJSW0 181 1764 mdash J Craig Venter InstituteHaemophilus paraphrohaemolyticusHK411a AJMU0 202 2025 mdash J Craig Venter InstituteHaemophilus sp oral taxon 851 strF0397a AGRK0 184 1809 mdash GCG-WUf

Histophilus somni 2336 CP000947 226 1980 374 Joint Genome InstituteHistophilus somni 129PT CP000436 201 1798 372 [51]Mannheimia succiniciproducensMBEL55E AE016827 231 2370 425 [52]Mannheimia haemolytica PHL213a AASA0 257 2695 411 [53]Pasteurella multocida subsp multocida str Pm70 AE004439 226 2012 404 [54]Pasteurella dagmatis ATCC 43325a ACZR0 225 2053 374 BCMg

aThe genomes of these speciesstrains are currently under scaffoldscontigs statusbStiftung Tieraerztliche Hochschule Hannover (STHH)cThe Broad Institute Genome Sequencing Platform (BIGSP)dUniversity of Washington Seattle Biomedical Research Institute (UW-SBRI)eCenter for Genomic Sciences Allegheny-Singer Research Institute (CGS ASRI)fGenome Sequencing Center (GSC) at Washington University (WashU) School of MedicinegBaylor College of Medicine (BCM)hWellcome Trust Sanger Institute (WTSI)


Actinobacillussensu stricto

Other Pasteurellaceae

Other Gammaproteobacteria

0250

Act pleuropneumonia ser 5b str L20Act pleuropneumoniae serovar 6 str FemoAct pleuropneumoniae serovar 1 str 4074Act pleuropneumoniae serovar 9 str CVJ13261Act pleuropneumoniae serovar 11 str 56153Act pleuropneumoniae serovar 12 str 1096Act pleuropneumonia ser 3 str JL03Act pleuropneumonia ser 7 str AP76Act pleuropneumoniae serovar 13 str N273Act pleuropneumoniae serovar 2 str S1536Act pleuropneumoniae serovar 2 str 4226Act pleuropneumoniae serovar 4 str M62Act pleuropneumoniae serovar 10 str D13039Actinobacillus capsulatusActinobacillus suis H91-0380Actinobacillus suis ATCC 33415Actinobacillus ureaeActinobacillus minorHaemophilus parasuisHaemophilus paraphrohaemolyticusHaemophilus parahaemolyticusBibersteinia trehalosi USDA-ARMannheimia haemolytica USDA-ARMannheimia haemolytica M42548Basfia succiniciproducensActinobacillus succinogenes 13Pasteurella bettyaePasteurella dagmatisPasteurella multocida HN06Pasteurella multocida 36950Aggregatibacter actinomycetemcomitansAggregatibacter aphrophilus NJAggregatibacter segnisHaemophilus haemolyticusHaemophilus influenzaePlesiomonas shigelloidesYersinia bercovieriSerratia odoriferaEnterobacter cloacae SCF1Dickeya zeae Ech1591Citrobacter freundiiShigella flexneriCronobacter turicensis z3032Vibrio vulnificus YJ016Klebsiella variicola At-22Pantoea ananatis LMG 5342Escherichia coli

126208128306860341306853561306862597306866939306869187165976058190149956306871346306855886302647429306858147306864716517480365407692352672592002491832514492353747759929664919922854919878784701671884723336194828866785242411915297781149213783849215028738331090737877494349174330825179286649377025149184999049195188449915175549141484049109435231128124125178842048992708949125365926059944637678593288937493378765502446511916

367QDDPTIQIVNQAQKAYVEN---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------V------------------V-------------------------------K------------------K----------A--------------------------------------------------------------T------------R-----A-K----------------A-------V----------A-------V-------R--A-------V-------R--A---------------------------------------K------------------K------------------K------------------K-----V----N-------H-----V----N--R--T-H-----V----N-------H-----V-V--M-------H-----V----N-------H-----V-V--N-------H-----V-V--N-------H-----V-V--N-------H-----V----L---D---R-----V-V--M-------H-----V----N--R----H-----V-V--N-------H

V----------------

402VVKGLPELTGLPVLSA----------------------------------------------------------------------------------------A---------------A---------------A---------------A---------------A---------------A---------------A---------------A---------------A---------------A----------S----A-------I-----Q-A---I---I-----Q-A---I----APSVA-M----I----ASSVA-MA---I---A--A--D-A---I-----------A---I-----------A---I------N----A-------IM-N--K-AK------I--N----A---I------N----A----------N----A----------N----A--------APSV-AMA---I----APSV-AMA---I----APSV-AMA---I----ASSVA-MA---I----APS-AAMA---I---F-Q-D-D-D-I-----FIQ-D-D-A-------YIQ-D-D-AD------FIQ-D-D-AN------FIQ-D-D-AE------FIQ-D-D-AK------YIQ-D-D-AK------FIQ-D-D-AT------FIQ-D-D-A-------FIQ-D-D-AK------FIQ-D-D-AT------YIQ-D-D-AK------

Figure 2 A partial sequence alignment of a 31015840-nucleotidase showing a 1-amino-acid insertion identified in allmembers ofActinobacillus sensustricto This insertion was not found in the homologues from any member of the genus Actinobacillus that was not part of the ldquosensu strictordquoclade or any other member of the Gammaproteobacteria Sequence information for a representative subset of the family Pasteurellaceae andthe classGammaproteobacteria is shown but unless otherwise indicated similar CSIs were detected in all members of the indicated group andnot detected in any other bacterial species in the top 250 BLAST hitsThe dashes (-) in the alignments indicate identity with the residue in thetop sequence GenBank identification (GI) numbers for each sequence are indicated in the second column Sequence information for otherCSIs specific to Actinobacillus sensu stricto are presented in Supplemental Figures 1-2 and their characteristics are summarized in Table 2(A)

Actinobacillus lignieresii the type species of the genus [24 78]However the original classification scheme for the genusActinobacillus led to the inclusion of a highly heterogeneousand polyphyletic grouping of species within the genus [12ndash14] An assemblage of Actinobacillus species closely relatedto Actinobacillus lignieresii has been recognized as Acti-nobacillus sensu stricto (ie the core members of the genusActinobacillus) in both our phylogenetic analysis (Figure 1)and past phylogenetic analyses [12ndash14 17] Differentiation ofActinobacillus sensu stricto from other Actinobacillus speciesand the modern criteria for placing novel species within thegenus Actinobacillus sensu stricto is heavily reliant on geneticand genomic criteria namely DNA-DNA hybridization

values 16S rRNA sequence similarity and other single genesequence comparisons [13 18]There are currently no knowndiscrete characteristics which are unique to Actinobacillusthat define the genus In this work we have completeda comprehensive comparative analysis of Pasteurellaceaegenomes in order to identify unique defining molecularsignatures for different genera within the family Pasteurel-laceae We have identified 3 CSIs which are unique definingmolecular signatures for the sequenced members of Acti-nobacillus sensu stricto (namely Actinobacillus capsulatusA pleuropneumoniae A suis and A ureae) An exampleof a CSI specific for Actinobacillus sensu stricto is shownin Figure 2 The CSI consists of a 1-amino-acid insertion


Table 2 Conserved signature indels specific for genera within the family Pasteurellaceae

Protein name Gene name GenBankidentifier Figure number Indel size Indel

positiona

(A) CSIs specific for Actinobacillus sensu stricto31015840-nucleotidase surE 126208128 Figure 2 1 aa ins 367ndash402GTP pyrophosphokinase relA 126207889 Sup Figure 1 1 aa ins 368ndash412Anaerobic glycerol-3-phosphate dehydrogenase subunit glpA 491834528 Sup Figure 2 1 aa ins 359ndash400

(B) CSIs specific for Haemophilus sensu strictoBiotin-protein ligase birA 144979005 Figure 3 6 aa del 138ndash178Aspartate ammonia-lyase aspA 145630289 Sup Figure 3 1 aa ins 34ndash75NAD(P) transhydrogenase subunit alpha pntA 145631394 Sup Figure 4 1 aa del 352ndash378Fumarate reductase subunit C frdC 301169552 Sup Figure 5 3 aa ins 31ndash89Hypothetical tRNArRNA methyltransferase mdash 145636352 Sup Figure 6 1 aa del 17ndash58Gamma-glutamyl kinase proB 145629980 Sup Figure 7 1 aa ins 197ndash253ACP phosphodiesterase acpD 68250119 Sup Figure 8 2 aa del 119ndash159

(C) CSIs specific for Pasteurella sensu strictoMenaquinone-specific isochorismate synthase menF 386834899 Figure 4 4 aa ins 29ndash86tRNA s(4)U8 sulfurtransferase thiI 15602400 Sup Figure 9 2 aa del 412ndash446FKBP-type peptidyl-prolyl cis-trans isomerase slyD 378775595 Sup Figure 10 2 aa del 151ndash188Aspartate-semialdehyde dehydrogenase asd 383311492 Sup Figure 11 1 aa del 173ndash245Lactate permease family transporter lldP 492154065 Sup Figure 12 2 aa ins 390ndash427Cell division protein ftsA ftsA 492155843 Sup Figure 13 1 aa ins 357ndash387

(D) CSIs specific for AggregatibacternhaC family sodiumproton antiporter nhaC 493769836 Figure 5(a) 3 aa ins 396ndash437Outer membrane protein omp 261866907 Sup Figure 14 4 aa del 25ndash64Multidrug transportermurJ murJ 365966332 Sup Figure 15 1 aa del 190ndash220NADH dehydrogenase nuoE 387120244 Sup Figure 16 1 aa ins 372ndash412

(E) CSIs specific forMannheimiaMethyl-galactoside ABC transporter substrate-binding protein mdash 472335016 Figure 5(b) 1 aa del 33ndash73UDP-N-acetylmuramoylalanyl-D-glutamatendash26-diaminopimelate ligase murE 472333011 Sup Figure 17 2 aa del 418ndash473Glutathione-regulated potassium-efflux protein kefC 472333189 Sup Figure 18 1 aa ins 504ndash531Glycerol-3-phosphate acyltransferase plsB 472334521 Sup Figure 19 2 aa del 214ndash252

in a conserved region of a 31015840-nucleotidase which is presentin all sequenced members of Actinobacillus sensu strictoand absent in all other sequenced GammaproteobacteriaSequence information for 2 other CSIs which are also uniquecharacteristics of the Actinobacillus sensu stricto clade ispresented in Supplemental Figures 1-2 available online athttpdxdoiorg1011552015198560 and their characteris-tics are briefly summarized in Table 2(A)

34 Molecular Signatures Specific for Haemophilus sensustricto The classification of novel species into the genusHaemophilus was initially based on phenotypic and bio-chemical properties most importantly the dependence ofgrowth on the presence of factor V and factor X in blood[13 78 79] As with Actinobacillus the classification ofHaemophilus on the basis of phenotypic and biochemicalproperties has led to the genus containing an extremelyheterogeneous group of species [12ndash14 32] Species from thegenus Haemophilus have undergone a number of transfers

and reclassifications [21ndash24 28] However the genus remainshighly polyphyletic (Figure 1) [17 19 28] The core mem-bers of the genus Haemophilus (namely Haemophilus sensustricto) consist of Haemophilus influenzae H aegyptius andH haemolyticus based on 16S rRNA sequence analysis [12ndash14 32] However phylogenetic analysis based on DNA-DNAhybridization and multilocus sequence analysis suggests thatH parainfluenzae and H pittmaniae are also members ofHaemophilus sensu stricto [30 80] Phylogenetic analysis ofrpoB infB and concatenated gene sets also suggest that[Pasteurella] pneumotropica and related isolates are closelyrelated to Haemophilus sensu stricto [15 16]

Our comparative analysis of Pasteurellaceae genomeshas led to the identification of 7 CSIs that are uniquecharacteristics of Haemophilus sensu stricto which consistsof Haemophilus influenzae H aegyptius H haemolyticus Hparainfluenzae H pittmaniae and [Pasteurella] pneumotrop-ica (Figure 1) One example of a CSI specific for the membersof Haemophilus sensu stricto shown in Figure 3 consists ofa 4-amino-acid deletion in a biotin-protein ligase which is


OtherHaemophilus

OtherPasteurellaceae


0250

Haemophilus sensu stricto

Haemophilus influenzae 221-21Haemophilus influenzae PittHHHaemophilus influenzae R3021Haemophilus influenzae R2866Haemophilus influenzae PittAAHaemophilus influenzae PittIIHaemophilus influenzae R2846Haemophilus influenzae 10810Haemophilus influenzae 7P49H1Haemophilus influenzae PittEEHaemophilus influenzae 86-028NPHaemophilus influenzae F3031Haemophilus influenzae CGSHiCZ412602Haemophilus influenzae 7P49H1Haemophilus influenzae 3655Haemophilus influenzae 6P18H1Haemophilus influenzae NT127Haemophilus influenzae KR494Haemophilus influenzae Rd KW20Haemophilus influenzae PittGGHaemophilus haemolyticus M21621Haemophilus haemolyticus HK386Haemophilus haemolyticus M19107Haemophilus haemolyticus M21127Haemophilus haemolyticus M19501Pasteurella pneumotropicaHaemophilus sp oral taxon 851Haemophilus parainfluenzae ATCC 33392Haemophilus parainfluenzae T3T1Haemophilus parainfluenzae HK262Haemophilus pittmaniaeHaemophilus sputorumHaemophilus paraphrohaemolyticusHaemophilus parahaemolyticusHaemophilus ducreyiHaemophilus parasuisHistophilus somniAggregatibacter segnisAggregatibacter actinomycetemcomitansAggregatibacter aphrophilusActinobacillus pleuropneumoniaeActinobacillus ureaeActinobacillus minorActinobacillus succinogenesBasfia succiniciproducensMannheimia haemolytica Pasteurella multocidaPasteurella dagmatisGallibacterium anatisCronobacter sakazakiiEdwardsiella ictaluriEnterobacter cloacaeErwinia tasmaniensisEscherichia coliKlebsiella pneumoniaePantoea vagansPhotorhabdus luminescensSerratia odorifera

1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458

138LSLVIGLAIAEVL----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------V------AF------------------------A------------A------------A----A-------TF----VA-VL--SF----VS-I---A-----VS-I---A-----VA-I---T-----VSVL---TF--S-L---T------T------VQA---S------VQA---S------VQS------S-I---S------S-I---S-----VA-I---S----TV-M--HRAI------M---DAI----VA-I---S-----V-M----T-----V-M---DT----AV-M-V-QA-------IVM----------IVM----------IVM----------IV---A-------IVM----------IV-----------IVM--T-----V-IV-----------IVM----

TELGISQAQNVQQAQNVQQAQQVEQTLNVPNEMGAVELDMYAELDMYVELDMYQAQQVEQAQQVEQAQGVKRKLGSDKSAGGKTVQNVKKQAGALRRTGVRTELNLHHELGAAQALGAAQRLGAEQQQGAPRKLGADQQLGAERALGADHRFGAGQRLGAE

173NVQVKWPNDILFDERKLGGILVE------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------G----------------------G----------------------G---------K------------G------------------M--EG---------G------------G-------I--------------G-----------K--------LSG-----------K--------LSG----------AK------V-LSG---------GA-------V-YQDA-MA--SLDDI-IK----VYYQGK------I-DI-I------YYQGK-M----I-DI-I-----VYYQGK-M----I-HI-I------YYQGK-M----I-HI-I------YYQGK-M----I-E-KL-----L-LFG---A-----GF---------VN----A-----GF---------VND---A-----GF---------VND---A------I-I------YYCSK-M----I--I-I------Y-CGK-M----I-DI-I-----VY-QGK-M----I-QTKL-----L-LHG---A---I-EINL-----L-LNG---A---I-DI-I------YYQGK-M----L--IGL-----V-LHG---A-------KL-----V-LNG---A-----S--L------WLNGK------I-Q-R------LYLHD---A-----G-K------LYLND---A-----G-R------LYLQD---A-----DIR-------YLND---A-----K-R------LYLQD---A-----Q-R-------YLQD---S-----D-R-------YLND---A-----RIR------LYL-DK--A-----E-R------LYLND---A-----

Figure 3 A partial sequence alignment of 14-dihydroxy-2-naphthoate octaprenyltransferase showing a 2-amino-acid insertion identified inall members of Haemophilus sensu stricto This insertion was not found in the homologues from any member of the genus Haemophilus thatwas not part of the ldquosensu strictordquo clade or any other member of the Gammaproteobacteria Sequence information for other CSIs specific toHaemophilus sensu stricto is presented in Supplemental Figures 3ndash8 and their characteristics are summarized in Table 2(B)

uniquely found in homologs from Haemophilus sensu strictoand absent in all other sequenced GammaproteobacteriaSequence information for 6 additional CSIs which are alsounique characteristics of Haemophilus sensu stricto is pre-sented in Supplemental Figures 3ndash8 and their characteris-tics are briefly summarized in Table 2(B) These CSIs andour phylogenetic trees (Figure 1) suggest that Haemophilus

influenzae H aegyptius H haemolyticus H parainfluenzaeH pittmaniae and [Pasteurella] pneumotropica share a closeevolutionary relationship and should all be considered mem-bers of Haemophilus sensu stricto Additionally these resultsalso suggest that [Pasteurella] pneumotropica is incorrectlyclassified as a member of the genus Pasteurella and should bereclassified as ldquoHaemophilus pneumotropicardquo


Pasteurellasensu stricto



0250

Pasteurella multocida 3480Pasteurella multocida HN06Pasteurella multocida Pm70Pasteurella multocida Anand1Pasteurella multocida X73Pasteurella multocida 2000Pasteurella multocida 93002Pasteurella multocida 67190Pasteurella multocida HB03Pasteurella multocida P1933Pasteurella multocida P52VACPasteurella multocidaPasteurella multocida RIIFPasteurella multocida 1500CPasteurella multocida PMTBPasteurella multocida 36950Pasteurella dagmatisPasteurella bettyaeBasfia succiniciproducensActinobacillus succinogenes 13Aggregatibacter actinomycetemcomitansAggregatibacter segnisGallibacterium anatis UMN179Histophilus somni 129PTHaemophilus parainfluenzae T3THaemophilus pittmaniaeHaemophilus influenzae Rd KW20Haemophilus sputorumHaemophilus parasuis SH0165Haemophilus ducreyi 35000HPActinobacillus minorActinobacillus ureaeActinobacillus suis H91-0380Actinobacillus pleuropneumoniaMannheimia haemolytica USDA-ARBibersteinia trehalosi USDA-ARMoritella sp PE36Grimontia sp AK16Plesiomonas shigelloidesPhotobacterium damselaeEscherichia sp TW09231Aliivibrio salmonicida LFI1238Vibrio fischeri ES114Yokenella regensburgeiYersinia bercovieriAliivibrio fischeriPhotobacterium sp AK15Klebsiella oxytoca

3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845

29WYAGTLGVMGPAYADFCVTIRSAFIE-----------T-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F-SKMQ--------------------F-SQVKSE----L----V--------F-NR-R-E----L----V----------TKEHSE------------------FFNR-R-E------------------FFNQQQ-E---A-----V-----AI--ISH-F-E---GL---KLT----A--I-TETESE-------S------------SQNLSE------------------L-SREQ-E--------------------SDVCSE---A-----------A--FVS-ERSE----L---QVH----A--YFT-EQ-E---ML---L-Q-------YFHTDH-E-T--L---K-D-------IL-EDE-E----L---Q-K-------YLQ-DE-E---AL---Q-------F-YLQ-DE-E---AL---Q---------YLQ-DE-E---AL---Q------------NEE-E----L---L-S-------YFS--Q-E----L---LVR--S-A--YV-QQKSE---A----R-L--S-SV-YLS-EQSE---A----L-V----SV-HISRER-E-T-A----L-Q--S-AV-YLSRQHSE---A----L-A----SA-YLSLQQSE---SL---K-S--S-AV-FLSQQRSE---A----LVM--S-AV-FLSQQRSE---A----LVM----SA-YLSRQQSE---AL---MVS----SA-YLSRQQSE-S--L---WL---S-AV-FLSQQRSE---A----LVM--S-AV-FLSRQRSE---A----L-M----SA-YLSL-QSE---SL---KVQ

QAEN-------------------------------------------------------------MD-

80DSQLCVFAGAGIVEGSIPLLEW------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Q-K-------------------QNRIRI-------A--------QNRIR--------A--V-----SNKIR--------A--V-----ADKIR-----------V-----ADKIH-----------V-----HQ--HL-------KE-QADE--QDYIRI----------------EN-VR-----------Q-VE--QQ-IR--------A--D--A--GHRIR--------A--Q--E--GNK-I-Y------A--E-QA--AN-ITFY-------K-D-QS--HN--TLY------AE-QADS--QN-VTLY------QD-E--S--QNCITLY-------E-E-QS--QNCITLY-------E-E-QS--RNRITLY-------E-E-QS--QNSITLY------S--D-QS--AKEML-Y------AE-E-ES--ENE-QL-------P--D-MS---NKVHL-------P--VAES--QN-VHL-------P--D-EA--GEE-HL-------P--D-SS---NVVRLY------R--D-EQ--GNK-HL-------P--E-SS--GNK-HL-------P--E-SS--GET-RLY------R--D-E----QWVNLY------A--D-E---GNK-HL-------P--E-SS--GEE-HL-------P--TADS--QHT-RLY------S--D-EQ--

Figure 4 A partial sequence alignment of Menaquinone-specific isochorismate synthase showing a 4-amino-acid insertion identified in allmembers of Pasteurella sensu stricto This insertion was not found in the homologues from any member of the genus Pasteurella that was notpart of the ldquosensu strictordquo clade or any other member of theGammaproteobacteria Sequence information for other CSIs specific to Pasteurellasensu stricto is presented in Supplemental Figures 9ndash13 and their characteristics are summarized in Table 2(C)

35 Molecular Signatures Specific for Pasteurella sensu strictoThe genus Pasteurella is highly heterogeneous and poly-phyletic (Figure 1) [13] Similar to the members of Acti-nobacillus bacterial isolates were originally classified asmembers of the genus Pasteurella based on growth fac-tor independent growth and phenotypic or biochemicalsimilarity to Pasteurella multocida the type species of thegenus [78 81] The monophyletic clusters of Pasteurellaspecies that branch with Pasteurella multocida are consid-ered the core members of the genus (namely Pasteurellasensu stricto) [9 13 16 17] Our comparative analysis ofPasteurellaceae genomes has led to the identification of 6CSIs which are unique characteristics for the sequencedmembers of Pasteurella sensu stricto (namely Pasteurellamultocida and P dagmatis) An example of a CSI uniquely

found in the sequenced members of Pasteurella sensu strictoconsisting of a 4-amino-acid insertion in a conserved regionof Menaquinone-specific isochorismate synthase is shownin Figure 4 This CSI is only found in the sequencedmembers of Pasteurella sensu stricto and is absent fromall other sequenced Gammaproteobacteria Partial sequencealignments for 5 additional CSIs which are also uniquecharacteristics of Pasteurella sensu stricto are presented inSupplemental Figures 9ndash13 and their characteristics arebriefly summarized in Table 2(C)

36 Molecular Signatures Specific for the Genera Aggregatibac-ter or Mannheimia The genus Aggregatibacter was proposedas a novel taxonomic classification for a monophyletic clusterof Actinobacillus and Haemophilus species which branched


Aggregatibacter



0250

Aggregatibacter segnis ATCC 33393Aggregatibacter aphrophilus NJAggregatibacter aphrophilus F0387Agg actinomycetemcomitans D7S-1Agg actinomycetemcomitans serotype d str I63BAgg actinomycetemcomitans serotype b str SCC1398Agg actinomycetemcomitans serotype c str SCC2302Agg actinomycetemcomitans serotype c str AAS4AAgg actinomycetemcomitans serotype b str I23CAgg actinomycetemcomitans serotype b str SCC4092Agg actinomycetemcomitans serotype str D18P1Agg actinomycetemcomitans serotype c str D17P-2Agg actinomycetemcomitans serotype e str SC1083Agg actinomycetemcomitans serotype e str SCC393Agg actinomycetemcomitans RhAA1Agg actinomycetemcomitans D11S-1Agg actinomycetemcomitans ANH9381Agg actinomycetemcomitans D17P-3Agg actinomycetemcomitans Y4Avibacterium paragallinarumGallibacterium anatis UMN179Pasteurella pneumotropicaPasteurella dagmatisActinobacillus succinogenes 13Actinobacillus capsulatusActinobacillus minorPasteurella multocida 36950Pasteurella dagmatisPasteurella bettyaeActinobacillus ureaeBibersteinia trehalosi USDA-ARHaemophilus parasuis SH0165Haemophilus sputorumSuccinivibrionaceae bacteriumPsychromonas sp CNPT3Shewanella denitrificans OS217Grimontia hollisaeVibrio harveyiAeromonas molluscorumVibrio sp Ex25

3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622

396TSWGTFGIMLPIAAAIASHAMP-----------------A--AA-----------------A--AA---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H---------------------H-----------------AN-A----------------M-AN-D---------------SM-TN-A------------------AN-A------------------TN-A---------------GM-INVDT------------G-SM-VNSD------------------ANTA------------------AN-A------------------VN-A---------------GM-INVDT---------------M-AN-E----------------M-AN-E-------A-----G-SM-M-SE---------------T--IN-NS-------------GDM-GATDV--------------DM-MGSDS-------------GDL-GATDI-------------GDM-GATDV-----------L-GDM-AASEI-------------GDM-GATDL

GSV------------------------------------------------------

437EFMLPCLSAVMAGAVCG-L----------------L------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------L---------------AL----------------L--A-------------L----------------LL--------------GLLI--M----------NLII--------------L----------------L----------------LL--------------GLLI--M----------ALL--S-----------ALL--------------ALII---A---S------LLI-------------A----M----L--S-F-TM---M----L----F-AL---M----L----F-AL---M----L----F-SM---M----L----F-AL---M----L----F-

(a)

Mannheimia



0250

Mannheimia haemolytica USDA-ARS-USMARC-183Mannheimia haemolytica D171Mannheimia haemolytica serotype A2 str OVINEMannheimia haemolytica D35Mannheimia haemolytica USMARC_2286Mannheimia haemolytica serotype 6 str H23Mannheimia haemolytica M42548Mannheimia haemolytica D174Mannheimia haemolytica D153Mannheimia haemolytica D38Mannheimia haemolytica MhBrain2012Mannheimia haemolytica MhSwine2000Mannheimia haemolytica D193Mannheimia varigena USDA-ARS-USMARC-1312Mannheimia varigena USDA-ARS-USMARC-1296Mannheimia varigena USDA-ARS-USMARC-1261Mannheimia varigena USDA-ARS-USMARC-1388Mannheimia granulomatisBibersteinia trehalosi USDA-ARHaemophilus parasuis SH0165Haemophilus paraphrohaemolyticusHaemophilus sputorumActinobacillus minorActinobacillus suis H91-0380Mannheimia succiniciproducensGallibacterium anatis UMN179Aggregatibacter actinomycetemcomitansHaemophilus parainfluenzae T3THaemophilus influenzae 86-028NErwinia amylovoraKlebsiella oxytocaYokenella regensburgeiTolumonas auensis DSM 9187Citrobacter rodentium ICC168Escherichia coli

4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822

33YKYDDNFMALMRKEIEKEGKTQK-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------QA---------------------QA---------------------QA---------------------QA---------------------AN---N------S------VQ-AARM---------S---Q-----V-VVG------------------AAQH---------S------N--A-ALN------------------A-NL----------------D--ANQL----------------D--ATNL---------S------N-DAEKVE--------S------Q-NAEQLQ--------S------D--A-ALG--------S------D--A-VVG--------SMV--D----A-NSP--------SVV--A---D--SAP--------SVV--A---DA-ASP--------SAV--A-----DQYP--------SVV--A--ADA-AAP--------SVV--A--QDA-AAP

EGDDDDDGNGGGDDDDD

73VELLMNDSQNTQSIQNDQ-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Q-----------------Q-----------------Q-----------------Q-----------------N--------A--------N--L-----A-----N--D--------A-----------------------------------A--------------------------K-----------------Q--------A-------IK--------A--------K--------A-------I---------A-------IK--------A--------Q--------S--T-----Q--------D--K----IQ--------D--K----IK--------D--K-----Q--------D--K-----Q--------D--K----

(b)

Figure 5 A partial sequence alignment of (a) a nhaC family sodiumproton antiporter containing a 3-amino-acid insertion specific forall sequenced species of the genus Aggregatibacter (b) a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specific for all sequenced species of the genus Mannheimia In each case the identified CSIs were only found in thesequencedmembers of the generaAggregatibacter orMannheimia and were absent from all other sequencedGammaproteobacteria Sequenceinformation for other CSIs specific to Aggregatibacter or Mannheimia is presented in Supplemental Figures 14ndash19 and their characteristicsare summarized in Tables 2(D) and 2(E)


distinctly from the ldquosensu strictordquomembers of their respectiveclades [21] Similarly the genus Mannheimia was proposedas a novel classification for the Pasteurella Haemolyticacomplex which did not branch with Pasteurella sensu strictoin phylogenetic trees [7] Currently other than branching inphylogenetic trees or relatedness in DNA-DNA hybridiza-tion studies the members of the genera Aggregatibacter orMannheimia do not share any single unique or definingbiochemical or molecular characteristic that can differentiatethem from all other bacteria [5 82]

In this study we have identified 4 CSIs that are uniquemolecular characteristics shared by all sequenced speciesof the genus Aggregatibacter and another 4 CSIs which areuniquely found in all sequenced members of the genusMannheimia Examples of CSIs specific to the sequencedmembers of Aggregatibacter and Mannheimia are shown inFigure 5 A partial sequence alignment of a nhaC familysodiumproton antiporter containing a 3-amino-acid inser-tion specific for all sequenced species of the genus Aggregati-bacter is shown in Figure 5(a) and a partial sequence align-ment of a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specificfor all sequenced species of the genus Mannheimia is shownin Figure 5(b) In each case the identified CSIs were onlyfound in the sequenced members of the genera Aggre-gatibacter or Mannheimia and were absent from all othersequenced Gammaproteobacteria Partial sequence align-ments additional CSIs specific for the genera AggregatibacterorMannheimia are provided in Supplemental Figures 14ndash19and their characteristics are summarized in Tables 2(D)-2 (E) These CSIs are the first discrete molecular charac-teristics which are unique for the genera Aggregatibacterand Mannheimia and support their observed monophylyin phylogenetic trees Additionally these CSIs could beuseful targets for the development of PCR based diagnosticassays for the genera Aggregatibacter andMannheimia whichamplify the CSI containing DNA segment using the con-served flanking regions of the CSIs [83 84]

4 Conclusion

The genera Actinobacillus Haemophilus and Pasteurellawithin the family Pasteurellaceae are known to exhibitextensive polyphyletic branchingWe have utilizedmolecularsignatures and phylogenetic analyses to clarify the taxonomicboundary of these genera We have been able to identifylarge clusters of Actinobacillus Haemophilus and Pasteurellaspecies which represent the ldquosensu strictordquo members of thesegenera We have identified 3 7 and 6 unique molecularsignatures which are specifically shared by the members ofthe genera Actinobacillus sensu stricto Haemophilus sensustricto and Pasteurella sensu stricto respectively The groupspecificity of the molecular signatures we have identified inthis work is summarized in Figure 6 and their characteristicsare briefly summarized in Table 2 Our comparative genomicanalyses have not come across any CSIs that were uniquecharacteristics of all sequenced members of the genera Acti-nobacillus Haemophilus or Pasteurella as currently definedsuggesting that the members of these genera that do not

7 CSIs specific for Haemophilus sensu


4 CSIs specific for Aggregatibacter

Aggregatibacter (Table 2(D))

6 CSIs specific for Pasteurella sensu


3 CSIs specific for Actinobacillus sensu


4 CSIs specific for Mannheimia

Mannheimia

(Table 2(E))

stricto (Table 2(B))

stricto (Table 2(C))

stricto (Table 2(A))

Figure 6 A summary diagram depicting the distribution of identi-fied CSIs for genera within the family Pasteurellaceae

fall into the ldquosensu strictordquo clusters should not be consideredmembers of their respective genus

Examinations of phenotypic and biochemical character-istics do not provide a reliable means of assigning a novelisolate to the genera Actinobacillus Haemophilus and Pas-teurella [18] However based upon the CSIs described in thiswork it is now possible to demarcate the genera Actinobacil-lus sensu stricto Haemophilus sensu stricto and Pasteurellasensu stricto on the basis of the presence or absence of uniquemolecular signatures It is important to note that the currentanalysis of CSIs is limited to the currently available genomicsequence data and may show slight variance as additionalbacterial genomes are sequenced However earlier work onCSIs for other groups of bacteria provides evidence that theidentified CSIs have strong predictive value and will likely befound in other members of these groups as more species aresequenced and novel species are isolated [74 77 85 86] Theconserved nature of the sequence regions that contain theseCSIs in conjunctionwith their strong predictive valuemakesCSIs promising targets for the development of highly specificdiagnostic assays forActinobacillus sensu strictoHaemophilussensu stricto Pasteurella sensu stricto Aggregatibacter andMannheimia [83 84] Additionally further analysis of thesegenus specific CSIs should lead to the discovery of theirfunctional role in their respective organisms andmay provideimportant insights into novel distinguishing features of thesegroups of organisms


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Sohail Naushad and Mobolaji Adeolu contributed equally tothis work

References

[1] A C Parte ldquoLPSNmdashlist of prokaryotic names with standing innomenclaturerdquo Nucleic Acids Research vol 42 no 1 pp D613ndashD616 2014

[2] K Muehldorfer S Speck and G Wibbelt ldquoProposal of Vesper-tiliibacter pulmonis gen nov sp nov and two genomospecies asnew members of the family Pasteurellaceae isolated from Euro-pean batsrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 64 no 7 pp 2424ndash2430 2014

[3] S M Spinola M E Bauer and R S Munson Jr ldquoImmu-nopathogenesis of Haemophilus ducreyi infection (chancroid)rdquoInfection and Immunity vol 70 no 4 pp 1667ndash1676 2002

[4] H Christensen and M Bisgaard ldquoMolecular classification andits impact on diagnostics and understanding the phylogenyand epidemiology of selected members of Pasteurellaceoe ofveterinary importancerdquo Berliner und Munchener TierarztlicheWochenschrift vol 123 no 1-2 pp 20ndash30 2010

[5] N Noslashrskov-Lauritsen ldquoClassification identification and clin-ical significance of Haemophilus and Aggregatibacter specieswith host specificity for humansrdquoClinicalMicrobiology Reviewsvol 27 no 2 pp 214ndash240 2014

[6] B Henderson J M Ward and D Ready ldquoAggregatibacter(Actinobacillus) actinomycetemcomitans a triple Alowast periodon-topathogenrdquo Periodontology 2000 vol 54 no 1 pp 78ndash1052010

[7] Oslash Angen R Mutters D A Caugant J E Olsen and M Bis-gaard ldquoTaxonomic relationships of the [Pasteurella] haemolyt-ica complex as evaluated by DNA-DNA hybridizations and 16srRNA sequencing with proposal of Mannheimia haemolyticagen nov comb nov Mannheimia granulomatis comb novMannheimia glucosida sp nov Mannheimia ruminalis sp novand Mannheimia varigena sp novrdquo International Journal ofSystematic Bacteriology vol 49 no 1 pp 67ndash86 1999

[8] J T Bosse H Janson B J Sheehan et al ldquoActinobacillus pleu-ropneumoniae pathobiology and pathogenesis of infectionrdquoMicrobes and Infection vol 4 no 2 pp 225ndash235 2002

[9] B A Wilson and M Ho ldquoPasteurella multocida from zoonosisto cellular microbiologyrdquo Clinical Microbiology Reviews vol 26no 3 pp 631ndash655 2013

[10] S PohlReklassifizierung der gattungActinobacillus Brumpt 1910Haemophilus Winslow et al 1971 und Pasteurella Trevisan 1887anhand phanotypischer undmolekularer daten insbesondere derDNS-verwandtschaften bei DNS DNS-hybridisierung in vitround vorschlag einer neuen familie Pasteurellaceae [Inaug Diss]Philipps-Universitat Marburg Germany 1979

[11] W Mannheim S Pohl and R Hollander ldquoOn the taxonomy ofActinobacillus Haemophilus and Pasteurella DNA base com-position respiratory quinones and biochemical reactions ofrepresentative collection cultures (authorrsquos transl)rdquo Zentralblattfur Bakteriologie A vol 246 no 4 pp 512ndash540 1979

[12] F E Dewhirst B J Paster I Olsen and G J Fraser ldquoPhylogenyof 54 representative strains of species in the family Pasteurel-laceae as determined by comparison of 16S rRNA sequencesrdquoJournal of Bacteriology vol 174 no 6 pp 2002ndash2013 1992

[13] I Olsen F E Dewhirst B J Paster and H J Busse ldquoFamily IPasteurellaceaerdquo in Bergeyrsquos Manual of Systematic BacteriologyD J Brenner N R Krieg G M Garrity and J T Staley Edsvol 2 pp 851ndash856 Springer New York NY USA 2nd edition2005

[14] F E Dewhirst B J Paster I Olsen and G J Fraser ldquoPhy-logeny of the Pasteurellaceae as determined by comparisonof 16S ribosomal ribonucleic acid sequencesrdquo Zentralblatt furBakteriologie vol 279 no 1 pp 35ndash44 1993

[15] H Christensen P Kuhnert J E Olsen and M BisgaardldquoComparative phylogenies of the housekeeping genes atpD infBand rpoB and the 16S rRNA gene within the PasteurellaceaerdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 54 no 5 pp 1601ndash1609 2004

[16] B Korczak H Christensen S Emler J Frey and P Kuhn-ert ldquoPhylogeny of the family Pasteurellaceae based on rpoBsequencesrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 54 no 4 pp 1393ndash1399 2004

[17] P Kuhnert and B M Korczak ldquoPrediction of whole-genomeDNA-DNA similarity determination of G + C content andphylogenetic analysis within the family Pasteurellaceae by mul-tilocus sequence analysis (MLSA)rdquoMicrobiology vol 152 no 9pp 2537ndash2548 2006

[18] H Christensen P Kuhnert H J Busse W C Frederiksen andM Bisgaard ldquoProposed minimal standards for the descriptionof genera species and subspecies of the Pasteurellaceaerdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 57 no 1 pp 166ndash178 2007

[19] M P D Bonaventura E K Lee R DeSalle and P J PlanetldquoA whole-genome phylogeny of the family PasteurellaceaerdquoMolecular Phylogenetics and Evolution vol 54 no 3 pp 950ndash956 2010

[20] H S Naushad and R S Gupta ldquoMolecular signatures (con-served indels) in protein sequences that are specific for the orderPasteurellales and distinguish two of its main cladesrdquo Antonievan Leeuwenhoek vol 101 no 1 pp 105ndash124 2012

[21] N Noslashrskov-Lauritsen and M Kilian ldquoReclassification of Acti-nobacillus actinomycetemcomitans Haemophilus aphrophilusHaemophilus paraphrophilus and Haemophilus segnis as Aggre-gatibacter actinomycetemcomitans gen nov comb nov Aggre-gatibacter aphrophilus comb nov and Aggregatibacter seg-nis comb nov and emended description of Aggregatibacteraphrophilus to include V factor-dependent and V factor-independent isolatesrdquo International Journal of Systematic andEvolutionary Microbiology vol 56 no 9 pp 2135ndash2146 2006

[22] P J Blackall H Christensen T Beckenham L L Blackalland M Bisgaard ldquoReclassification of Pasteurella gallinarum[Haemophilus] paragallinarum Pasteurella avium and Pas-teurella volantium as Avibacterium gallinarum gen nov combnov Avibacterium paragallinarum comb nov Avibacteriumavium comb nov and Avibacterium volantium comb novrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 55 no 1 pp 353ndash362 2005

[23] Oslash Angen P Ahrens P Kuhnert H Christensen and RMutters ldquoProposal of Histophilus somni gen nov sp novfor the three species incertae sedis lsquoHaemophilus somnusrsquolsquoHaemophilus agnirsquo and lsquoHistophilus ovisrsquordquo International Journalof Systematic and Evolutionary Microbiology vol 53 no 5 pp1449ndash1456 2003


[24] S Pohl H U Bertschinger W Frederiksen andWMannheimldquoTransfer of Haemophilus pleuropneumoniae and the Pas-teurella haemolytica-like organism causing porcine necroticpleuropneumonia to the genus Actinobacillus (Actinobacilluspleuropneumoniae comb nov) on the basis of phenotypicand deoxyribonucleic acid relatednessrdquo International Journal ofSystematic Bacteriology vol 33 no 3 pp 510ndash514 1983

[25] H Christensen M Bisgaard A M Bojesen R Mutters and JE Olsen ldquoGenetic relationship among avian isolates classifiedas Pasteurella haemolytica rsquoActinobacillus salpingitidisrsquo or Pas-teurella anatis with proposal of Gallibacterium anatis gen novcomb nov and description of additional genomospecies withGallibacterium gen novrdquo International Journal of Systematic andEvolutionary Microbiology vol 53 no 1 pp 275ndash287 2003

[26] P J Blackall A M Bojesen H Christensen and M BisgaardldquoReclassification of [Pasteurella] trehalosi as Bibersteinia tre-halosi gen nov comb novrdquo International Journal of Systematicand Evolutionary Microbiology vol 57 no 4 pp 666ndash674 2007

[27] R Mutters S Pohl and W Mannheim ldquoTransfer of Pasteurellaureae Jones 1962 to the genus Actinobacillus Brumpt 1910 Acti-nobacillus ureae comb novrdquo International Journal of SystematicBacteriology vol 36 no 2 pp 343ndash344 1986

[28] H Christensen and P Kuhnert ldquoInternational committee onsystematics of prokaryotes Subcommittee on the taxonomyof Pasteurellaceae Minutes of the meetings 25 August 2011Elsinore Denmarkrdquo International Journal of Systematic andEvolutionary Microbiology vol 62 no 1 pp 257ndash258 2012

[29] J Hedegaard H Okkels B Bruun M Kilian K KMortensen and N Noslashrskov-Lauritsen ldquoPhylogeny of thegenus Haemophilus as determined by comparison of partialinfB sequencesrdquo Microbiology vol 147 no 9 pp 2599ndash26092001

[30] N Noslashrskov-Lauritsen B Bruun and M Kilian ldquoMultilocussequence phylogenetic study of the genus Haemophilus withdescription of Haemophilus pittmaniae sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 55 no1 pp 449ndash456 2005

[31] V Cattoir O Lemenand J-L Avril and O Gaillot ldquoThesodA gene as a target for phylogenetic dissection of the genusHaemophilus and accurate identification of human clinicalisolatesrdquo International Journal ofMedicalMicrobiology vol 296no 8 pp 531ndash540 2006

[32] M Kilian ldquoGenus III Haemophilusrdquo in Bergeyrsquos Manual ofSystematic Bacteriology D J BrennerN RKriegGMGarrityand J T Staley Eds vol 2 pp 883ndash904 Springer New YorkNY USA 2nd edition 2005

[33] N Noslashrskov-Lauritsen B Bruun C Andersen and M KilianldquoIdentification of haemolyticHaemophilus species isolated fromhuman clinical specimens and description of Haemophilussputorum sp novrdquo International Journal of Medical Microbiol-ogy vol 302 no 2 pp 78ndash83 2012

[34] R D Fleischmann M D Adams O White et al ldquoWhole-genome random sequencing and assembly of Haemophilusinfluenzae Rdrdquo Science vol 269 no 5223 pp 496ndash512 1995

[35] A R Wattam D Abraham O Dalay et al ldquoPATRIC the bac-terial bioinformatics database and analysis resourcerdquo NucleicAcids Research vol 42 no 1 pp D581ndashD591 2014

[36] NCBI NCBI Genome Database 2014 httpwwwncbinlmnihgovgenome

[37] NCBI NCBI Nucleotide Database 2014 httpwwwncbinlmnihgovnuccore

[38] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[39] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[40] S J Foote J T Bosse A B Bouevitch P R Langford N MYoung and J H E Nash ldquoThe complete genome sequence ofActinobacillus pleuropneumoniae L20 (serotype 5b)rdquo Journal ofBacteriology vol 190 no 4 pp 1495ndash1496 2008

[41] Z Xu Y Zhou L Li et al ldquoGenome biology of Actinobacilluspleuropneumoniae JL03 an isolate of serotype 3 prevalent inChinardquo PLoS ONE vol 3 no 1 Article ID e1450 2008

[42] M P Di Bonaventura R DeSalle M Pop et al ldquoCom-plete genome sequence of Aggregatibacter (Haemophilus)aphrophilus NJ8700rdquo Journal of Bacteriology vol 191 no 14 pp4693ndash4694 2009

[43] C Chen W Kittichotirat Y Si and R Bumgarner ldquoGenomesequence of Aggregatibacter actinomycetemcomitans serotype cstrain D11S-1rdquo Journal of Bacteriology vol 191 no 23 pp 7378ndash7379 2009

[44] C Chen W Kittichotirat W Chen J S Downey Y Siand R Bumgarner ldquoGenome sequence of naturally competentAggregatibacter actinomycetemcomitans serotype a strain D7S-1rdquo Journal of Bacteriology vol 192 no 10 pp 2643ndash2644 2010

[45] T J Johnson C Fernandez-Alarcon AM Bojesen L KNolanD W Trampel and T Seemann ldquoComplete genome sequenceof Gallibacterium anatis strain UMN179 isolated from a layinghen with peritonitisrdquo Journal of Bacteriology vol 193 no 14 pp3676ndash3677 2011

[46] I K Jordan A B Conley I V Antonov et al ldquoGenomesequences for five strains of the emerging pathogenHaemophilus haemolyticusrdquo Journal of Bacteriology vol193 no 20 pp 5879ndash5880 2011

[47] F R Strouts P Power N J Croucher et al ldquoLineage-specificvirulence determinants of Haemophilus influenzae biogroupaegyptiusrdquo Emerging Infectious Diseases vol 18 no 3 pp 449ndash457 2012

[48] J S Hogg F Z Hu B Janto et al ldquoCharacterization and mod-eling of the Haemophilus influenzae core and supragenomesbased on the complete genomic sequences of Rd and 12 clinicalnontypeable strainsrdquo Genome Biology vol 8 no 6 article R1032007

[49] A Harrison D W Dyer A Gillaspy et al ldquoGenomic sequenceof an otitis media isolate of nontypeable Haemophilus influen-zae comparative study with H influenzae serotyped strainKW20rdquo Journal of Bacteriology vol 187 no 13 pp 4627ndash46362005

[50] M Yue F Yang J Yang et al ldquoComplete genome sequence ofHaemophilus parasuisSH0165rdquo Journal of Bacteriology vol 191no 4 pp 1359ndash1360 2009

[51] J F Challacombe A J Duncan T S Brettin et al ldquoCompletegenome sequence of Haemophilus somnus (Histophilus somni)strain 129Pt and comparison to Haemophilus ducreyi 35000HPand Haemophilus influenzae Rdrdquo Journal of Bacteriology vol189 no 5 pp 1890ndash1898 2007

[52] S H Hong J S Kim S Y Lee et al ldquoThe genome sequenceof the capnophilic rumen bacteriumMannheimia succinicipro-ducensrdquo Nature Biotechnology vol 22 no 10 pp 1275ndash12812004


[53] J Gioia X Qin H Jiang et al ldquoThe genome sequence ofMannheimia haemolytica A1 insights into virulence naturalcompetence and Pasteurellaceae phylogenyrdquo Journal of Bacte-riology vol 188 no 20 pp 7257ndash7266 2006

[54] B JMayQ Zhang L L LiM L Paustian T SWhittam andVKapur ldquoComplete genomic sequence of Pasteurella multocidaPm70rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 6 pp 3460ndash3465 2001

[55] R C Edgar ldquoSearch and clustering orders of magnitude fasterthan BLASTrdquo Bioinformatics vol 26 no 19 Article ID btq461pp 2460ndash2461 2010

[56] K Katoh and D M Standley ldquoMAFFT multiple sequencealignment software version 7 improvements in performanceand usabilityrdquo Molecular Biology and Evolution vol 30 no 4pp 772ndash780 2013

[57] M N Price P S Dehal and A P Arkin ldquoFastTree 2mdashapproximatelymaximum-likelihood trees for large alignmentsrdquoPLoS ONE vol 5 no 3 Article ID e9490 2010

[58] S Whelan and N Goldman ldquoA general empirical modelof protein evolution derived from multiple protein familiesusing a maximum-likelihood approachrdquoMolecular Biology andEvolution vol 18 no 5 pp 691ndash699 2001

[59] R S Gupta ldquoIdentification of conserved indels that are usefulfor classification and evolutionary studiesrdquo in Methods inMicrobiology vol 41 pp 153ndash182 Academic Press 2014

[60] F Jeanmougin J DThompson M Gouy D G Higgins and TJ Gibson ldquoMultiple sequence alignment with Clustal Xrdquo Trendsin Biochemical Sciences vol 23 no 10 pp 403ndash405 1998

[61] E Stackebrandt and J Ebers ldquoTaxonomic parameters revisitedtarnished gold standardsrdquoMicrobiology Today vol 33 no 4 p152 2006

[62] K T Konstantinidis and E Stackebrandt ldquoDefining taxonomicranksrdquo inThe Prokaryotes pp 229ndash254 Springer 2013

[63] R J Redfield W A Findlay J Bosse J S Kroll A D SCameron and J H E Nash ldquoEvolution of competence andDNA uptake specificity in the Pasteurellaceaerdquo BMC Evolution-ary Biology vol 6 article 82 2006

[64] A Rokas B I Williams N King and S B Carroll ldquoGenome-scale approaches to resolving incongruence in molecular phy-logeniesrdquo Nature vol 425 no 6960 pp 798ndash804 2003

[65] D Wu P Hugenholtz K Mavromatis et al ldquoA phylogeny-driven genomic encyclopaedia of Bacteria and ArchaeardquoNature vol 462 no 7276 pp 1056ndash1060 2009

[66] P Yilmaz L W Parfrey P Yarza et al ldquoThe SILVA andrsquosll-species living tree project (LTP)rsquo taxonomic frameworksrdquoNucleic Acids Research 2013

[67] B Gao RMohan and R S Gupta ldquoPhylogenomics and proteinsignatures elucidating the evolutionary relationships among theGammaproteobacteriardquo International Journal of Systematic andEvolutionary Microbiology vol 59 no 2 pp 234ndash247 2009

[68] A M Cutino-Jimenez M Martins-Pinheiro W C Lima AMartın-Tornet O G Morales and C F M Menck ldquoEvolution-ary placement of Xanthomonadales based on conserved proteinsignature sequencesrdquo Molecular Phylogenetics and Evolutionvol 54 no 2 pp 524ndash534 2010

[69] H S Naushad and R S Gupta ldquoPhylogenomics and molecularsignatures for species from the plant pathogen-containing orderXanthomonadalesrdquo PLoS ONE vol 8 no 2 Article ID e552162013

[70] R S Gupta ldquoApplications of conserved indels for understand-ing microbial phylogenyrdquo in Molecular Phylogeny of Microor-ganisms A Oren and R T Papke Eds pp 135ndash150 CaisterAcademic Press Norfolk UK 2010

[71] H S Naushad B Lee and R S Gupta ldquoConserved signatureindels and signature proteins as novel tools for understandingmicrobial phylogeny and systematics Identification of molecu-lar signatures that are specific for the phytopathogenic generaDickeya Pectobacterium and Brenneriardquo International Journalof Systematic and Evolutionary Microbiology vol 64 no 2 pp366ndash383 2014

[72] R S Gupta ldquoProtein phylogenies and signature sequences areappraisal of evolutionary relationships among archaebacteriaeubacteria and eukaryotesrdquo Microbiology and Molecular Biol-ogy Reviews vol 62 no 4 pp 1435ndash1491 1998

[73] A Rokas and PW H Holland ldquoRare genomic changes as a toolfor phylogeneticsrdquo Trends in Ecology and Evolution vol 15 no11 pp 454ndash459 2000

[74] R S Gupta and R Lali ldquoMolecular signatures for the phylumAquificae and its different clades Proposal for division ofthe phylum Aquificae into the emended order Aquificalescontaining the familiesAquificaceae andHydrogenothermaceaeand a new order Desulfurobacteriales ord nov containing thefamily Desulfurobacteriaceaerdquo Antonie van Leeuwenhoek vol104 no 3 pp 349ndash368 2013

[75] R S Gupta S Mahmood and M Adeolu ldquoA phylogenomicand molecular signature based approach for characterizationof the phylum spirochaetes and its major clades proposal fora taxonomic revision of the phylumrdquo Frontiers in Microbiologyvol 4 article 217 2013

[76] M Adeolu and R S Gupta ldquoA phylogenomic and molecularmarker based proposal for the division of the genus Borreliainto two genera the emended genus Borrelia containing onlythe members of the relapsing fever Borrelia and the genusBorreliella gen nov containing the members of the Lymedisease Borrelia (Borrelia burgdorferi sensu lato complex)rdquoAntonie van Leeuwenhoek vol 105 no 6 pp 1049ndash1072 2014

[77] V Bhandari and R S Gupta ldquoMolecular signatures for thephylum (class) Thermotogae and a proposal for its divisioninto three orders (Thermotogales Kosmotogales ord Nov andPetrotogales ord Nov) containing four families (Thermoto-gaceae Fervidobacteriaceae famNovKosmotogaceae famNovand Petrotogaceae fam Nov) and a new genus Pseudother-motoga gen Nov with five new combinationsrdquo Antonie vanLeeuwenhoek International Journal of General and MolecularMicrobiology vol 105 no 1 pp 143ndash168 2014

[78] I Olsen ldquoRecent approaches to the chemotaxonomy ofthe Actinobacillus-Haemophilus-Pasteurella group (family Pas-teurellaceae)rdquo Oral Microbiology and Immunology vol 8 no 6pp 327ndash336 1993

[79] N Hayashimoto M Ueno A Tkakura and T Itoh ldquoBiochem-ical characterization and phylogenetic analysis based on 16SrRNA sequences for V-factor dependent members of Pasteurel-laceae derived from laboratory ratsrdquo Current Microbiology vol54 no 6 pp 419ndash423 2007

[80] R Mutters W Frederiksen and W Mannheim ldquoTaxonomy ofthe grouprdquo in Pasturella and Pasteurellosis C Adlam and J MRutter Eds pp 3ndash34 Academic Press London UK 1989

[81] K P Snipes and E L Biberstein ldquoPasteurella testudinis sp nova parasite of desert tortoises (Gopherus agassizi)rdquo InternationalJournal of Systematic Bacteriology vol 32 no 2 pp 201ndash2101982


[82] Oslash Angen P Ahrens and M Bisgaard ldquoPhenotypic and geno-typic characterization of Mannheimia (Pasteurella) haemolyt-ica-like strains isolated from diseased animals in DenmarkrdquoVeterinary Microbiology vol 84 no 1-2 pp 103ndash114 2002

[83] N Z Ahmod R S Gupta and H N Shah ldquoIdentificationof a Bacillus anthracis specific indel in the yeaC gene anddevelopment of a rapid pyrosequencing assay for distinguishingB anthracis from the B cereus grouprdquo Journal of MicrobiologicalMethods vol 87 no 3 pp 278ndash285 2011

[84] S Y Wong A Paschos R S Gupta and H E Schell-horn ldquoInsertiondeletion-based approach for the detection ofEscherichia coliO157H7 in freshwater environmentsrdquo Environ-mental Science amp Technology vol 48 no 19 pp 11462ndash114702014

[85] B Gao and R S Gupta ldquoPhylogenetic framework and molecu-lar signatures for themain clades of the phylumActinobacteriardquoMicrobiology and Molecular Biology Reviews vol 76 no 1 pp66ndash112 2012

[86] M Howard-Azzeh L Shamseer H E Schellhorn and R SGupta ldquoPhylogenetic analysis and molecular signatures defin-ing a monophyletic clade of heterocystous cyanobacteria andidentifying its closest relativesrdquo Photosynthesis Research vol122 no 2 pp 171ndash185 2014


been transferred to the genus Aggregatibacter [21] thespecies [Haemophilus] paragallinarum [Pasteurella] galli-narum [Pasteurella] avium and [Pasteurella] volantium havebeen transferred to the genus Avibacterium [22] the species[Haemophilus] somnus and [Haemophilus] agni have beentransferred to the genus Histophilus [23] and the species[Pasteurella] haemolytica and [Pasteurella] granulomatis havebeen transferred to the genus Mannheimia [7] Additionallysome individual species within the genera ActinobacillusHaemophilus and Pasteurella that do not cluster with othermembers of their genus in phylogenetic trees have beenmoved or proposed to be moved to novel or neighbouringgenera (namely the transfer of the species [Haemophilus]pleuropneumoniae to the genus Actinobacillus [24] the trans-fer of the species [Pasteurella] anatis to the genus Gallibac-terium [25] the transfer of the species [Pasteurella] trehalosito the genus Bibersteinia [26] the transfer of the species [Pas-teurella] ureae to the genus Actinobacillus [27] and the pro-posed transfer of the species [Haemophilus] ducreyi to a novelgenus [28]) However despite these changes the classificationof the genera Actinobacillus Haemophilus and Pasteurellais still problematic and each genus continues to containmembers which exhibit polyphyletic branching [5 17ndash20]

Multiple studies have attempted to define a core group ofspecies which cluster around the nomenclatural type speciesofActinobacillus Haemophilus or Pasteurella as the only truemembers of these genera (ie sensu stricto) [13 15ndash17 29ndash31]but the taxonomy and phylogeny of these bacteria continue toremain inconclusive [20 32 33] Several methods have beenemployed for the demarcation of these genera however nosimplemethod or criterion is available that can clearly delimitthese genera It has been suggested that genome based studiesmay provide reliable means of clarifying the evolutionaryrelationships of these bacteria [33]

Since the availability of the first complete genomesequence of theHaemophilus influenzae [34] a large numberof genomes for the members of the family Pasteurellaceaehave become available in public databases [35 36] The avail-ability of these genomes provides us with an opportunity tocomplete comprehensive genome scale phylogenetic analysesof the family Pasteurellaceae These genome sequences havealso been utilized to carry out comparative genomic analysesto identifymolecular signatures (namely conserved signatureindels (CSIs) in various proteins) commonly shared by allor closely related subsets of species within the family Pas-teurellaceae On the basis of the molecular signatures iden-tified from comparative analyses of Pasteurellaceae genomesin conjunction with core genome based and multilocussequence based phylogenetic analyses we have identifiedsensu stricto clades of Actinobacillus Haemophilus and Pas-teurella that are supported by 3 7 and 6 unique molecularsignatures respectively We also report sets of molecularsignatures that are unique characteristics of the pathogencontaining genera Aggregatibacter andMannheimia

2 Methods

21 Multilocus Sequence Analysis Multilocus sequence anal-ysis was completed for members of the family Pasteurellaceae

using widely available nucleotide sequences of the 16S rDNAinfB (translation initiation factor IF-2) recN (DNA repairprotein) and rpoB (DNA-directed RNA polymerase subunitbeta) genes which have been used individually or as partof a set in a number of previous phylogenetic analyses ofthe family Pasteurellaceae [15ndash17 29 30] Gene sequences forthese four genes were obtained for 52 Pasteurellaceae strainsrepresenting a large majority of the known Pasteurellaceaespecies and 2 members of Vibrio cholerae from the NCBInucleotide database [37] Species which were missing one ofthese four genes or which did not have a gene sequence thatwas at least 50 of the length of the full gene were excludedfrom the analysis The four genes were individually alignedusing MUSCLE [38] and manually concatenated to create acombined dataset that contained 10 183 nucleotide long align-ments A maximum-likelihood tree based on 100 bootstrapreplicates of this alignment was constructed using MEGA60 [39] while employing maximum composite likelihoodsubstitution model

22 Pasteurellaceae Core Genome Phylogenetic Tree Aphylo-genetic tree of 76 Pasteurellaceae strains rooted using 7mem-bers of the family Vibrionaceae based on the core genomeof the family Pasteurellaceae was created for this study Thecore set of Pasteurellaceae proteins were identified usingthe UCLUST algorithm [55] to identify widely distributedprotein families with at least 30 sequence identity and50 sequence length Proteins families which were presentin less than 50 of the input genomes were excluded fromfurther analysis Potentially paralogous sequences (additionalproteins from the same organism in a single protein family)within the remaining protein families were also excludedfrom further analysis Each protein family was individuallyaligned using MAFFT 7 [56] Aligned amino acid positionswhich contained gaps in more than 50 of organisms wereexcluded from further analysis The remaining amino acidpositionswere concatenated to create a combined dataset thatcontained 128 080 amino acid long alignments An approx-imately maximum-likelihood tree based on this alignmentwas constructed using FastTree 2 [57] while employing theWhelan and Goldman substitution model [58]

23 Identification of Molecular Signatures (CSIs) for DifferentGenera of the Family Pasteurellaceae The detailed outlineof the process of identifying CSIs has been recently pub-lished [59] In brief Blastp searches were performed onall proteins from the genome of Haemophilus influenzaeF3047 [47] Ten to fifteen high scoring homologues thatwere present in Haemophilus other Pasteurellaceae andGammaproteobacteria species were retrieved and their mul-tiple sequence alignments were constructed using Clustal X183 [60] The alignments were visually inspected to identifyany conserved inserts or deletions (indels) that are restrictedto the particular clades of the family Pasteurellaceae whichare flanked on each side by at least 5-6 identicalconservedresidues in the neighbouring 30ndash40 amino acidsThe selectedsequences containing the indels and their flanking conservedregions were further evaluated by detailed Blastp searches

















Aggregatibacter





2975584-15995SH0165gx033ZJ0906MN-H12939SW114D74


HK411HK385



NM305202




Mannheimia


005

(a)













Mannheimia




01

(b)
























0250


126208128306860341306853561306862597306866939306869187165976058190149956306871346306855886302647429306858147306864716517480365407692352672592002491832514492353747759929664919922854919878784701671884723336194828866785242411915297781149213783849215028738331090737877494349174330825179286649377025149184999049195188449915175549141484049109435231128124125178842048992708949125365926059944637678593288937493378765502446511916


V----------------








positiona











OtherHaemophilus



0250



1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458











0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References










































































































Aggregatibacter





2975584-15995SH0165gx033ZJ0906MN-H12939SW114D74


HK411HK385



NM305202




Mannheimia


005

(a)













Mannheimia




01

(b)
























0250


126208128306860341306853561306862597306866939306869187165976058190149956306871346306855886302647429306858147306864716517480365407692352672592002491832514492353747759929664919922854919878784701671884723336194828866785242411915297781149213783849215028738331090737877494349174330825179286649377025149184999049195188449915175549141484049109435231128124125178842048992708949125365926059944637678593288937493378765502446511916


V----------------








positiona











OtherHaemophilus



0250



1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458











0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References
















































































































0250


126208128306860341306853561306862597306866939306869187165976058190149956306871346306855886302647429306858147306864716517480365407692352672592002491832514492353747759929664919922854919878784701671884723336194828866785242411915297781149213783849215028738331090737877494349174330825179286649377025149184999049195188449915175549141484049109435231128124125178842048992708949125365926059944637678593288937493378765502446511916


V----------------








positiona











OtherHaemophilus



0250



1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458











0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References





























































































positiona











OtherHaemophilus



0250



1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458











0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References



























































































OtherHaemophilus



0250



1449790051452696791449833933862635831452675481452710853862653973786963502298104021487156566805702831743206064622937622981040214498665822981206026009410754036511016272182501001793341954888386907988341948169341948545341948213517167265696223133325159690301156028385192842343517642359299234386390324387773709331513482198714661134605653156344702618676312517923051901512093225156772230415631529785681615109922543614121560216126091303933228977415693582523891813431128147218853230521870260815297283530818889837528549270265458











0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References






























































































0250


3868348993833108531560191833821798440438374851275374451275479751276043251275564251275564240169055740438473651276109051276308054458081537877488349215480249214591052425850152978460387121592493768552332289482113460763345428676494450864162722404947909522198714253315180649236715749183591240769182219015036747233329747016661149290354348849210049915106235841057044641846120969465659712279493874499491418549491562394494734248490215845


QAEN-------------------------------------------------------------MD-







Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References



























































































Aggregatibacter



0250


3154769492517926303533463683871199693479955533480054593480086214435428623480108524435449483480007093486563983479932093480008293597571502618681263659679063486541134291513495236740503322881215171679624921474031529787105174823294923540083787745824921474034921374904918323644701669192198713084947896934981410434704799539179388849164871213655509492636195262394622


GSV------------------------------------------------------


(a)

Mannheimia



0250


4723350165256572292613087185288223645264676634520881054810640885234350545234482515288249405288742225288772905288785235754446785754425435754421695754483506527557094701669882198706844919908324978139444923527034076933445242469833228995949169985834542969468249417490274190490205456493874411237809379283785958487492822


EGDDDDDGNGGGDDDDD


(b)





4 Conclusion











Mannheimia

(Table 2(E))












References





























































































4 Conclusion











Mannheimia

(Table 2(E))












References































































































References


























































































pasteurellaceae genera actinobacillus, haemophilus ... · researcharticle phylogenomic and...

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