# Phylogenomic and Molecular Demarcation of the Core Members of the PolyphyleticPasteurellaceae Genera Actinobacillus, Haemophilus, and Pasteurella

Authors: Sohail Naushad; Mobolaji Adeolu; Nisha Goel; Bijendra Khadka; Aqeel Al-Dahwi; Radhey S. Gupta
Journal: International Journal of Genomics
 (2015)

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## Abstract

The generaActinobacillus, Haemophilus,andPasteurella exhibit extensive polyphyletic branching in phylogenetic trees and do not represent coherent clusters of species. In this study, we have utilized molecular signatures identified through comparative genomic analyses in conjunction with genome based and multilocus sequence based phylogenetic analyses to clarify the phylogenetic and taxonomic boundary of these genera. We have identified large clusters ofActinobacillus, Haemophilus,andPasteurella species which represent the “sensu stricto” members of these genera. We have identified 3, 7, and 6 conserved signature indels (CSIs), which are specifically shared bysensu stricto members ofActinobacillus, Haemophilus,andPasteurella, respectively. We have also identified two different sets of CSIs that are unique characteristics of the pathogen containing generaAggregatibacter andMannheimia, respectively. It is now possible to demarcate the generaActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto on the basis of discrete molecular signatures. The other members of the generaActinobacillus, Haemophilus,andPasteurella that do not fall within the “sensu stricto” clades and do not contain these molecular signatures should be reclassified as other genera. The CSIs identified here also provide useful diagnostic targets for the identification of current and novel members of the indicated genera.


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## Body

## 1. Introduction

The familyPasteurellaceae, the single constituent family of the orderPasteurellales, represents a diverse group of commensal and pathogenic bacteria within the classGammaproteobacteria. The family currently contains 19 genera, some of which are particularly important human and animal pathogens [1, 2]. The generaHaemophilus contains species responsible for human bacteremia, pneumonia, acute bacterial meningitis, and the sexually transmitted disease chancroid [3–5];Aggregatibacter species have been implicated in juvenile periodontitis [6]; members of the generaMannheimia,Pasteurella, andActinobacillus have been implicated in the causation of shipping fever in cattle, fowl cholera, and pleuropneumonia in pigs, respectively [7–9].The familyPasteurellaceae was originally proposed as a higher level taxonomic grouping of the related pathogenic generaActinobacillus, Haemophilus,andPasteurella[10]. Classification of organisms into these three genera was primarily based on DNA G-C content and a handful of phenotypic traits [11]. The phenotypic traits were later found not to be characteristic of any single genus [12]. Consequently, the generaActinobacillus, Haemophilus,andPasteurella each exhibit extensive polyphyly in subsequent 16S rRNA based phylogenies [12, 13]. Additional studies based on individual or concatenated gene sets and DNA-DNA/rRNA-DNA hybridization also support the presence of extensive polyphyly within the generaActinobacillus, Haemophilus,andPasteurella [14–20].Extensive work has been undertaken to amend the classification of the generaActinobacillus, Haemophilus,andPasteurella[1, 5, 9, 18]. New genera have been created to house phylogenetically coherent clusters ofActinobacillus, Haemophilus,andPasteurella. The species [Actinobacillus]actinomycetemcomitans, [Haemophilus]aphrophilus, [Haemophilus]paraphrophilus, and [Haemophilus]segnis have been transferred to the genusAggregatibacter [21]; the species [Haemophilus]paragallinarum, [Pasteurella]gallinarum, [Pasteurella]avium, and [Pasteurella]volantium have been transferred to the genusAvibacterium[22]; the species [Haemophilus]somnus and [Haemophilus]agni have been transferred to the genusHistophilus[23]; and the species [Pasteurella]haemolytica and [Pasteurella]granulomatishave been transferred to the genusMannheimia[7]. Additionally, some individual species within the generaActinobacillus, Haemophilus,andPasteurella that do not cluster with other members of their genus in phylogenetic trees have been moved or proposed to be moved to novel or neighbouring genera (namely, the transfer of the species [Haemophilus]pleuropneumoniae to the genusActinobacillus[24], the transfer of the species [Pasteurella]anatis to the genusGallibacterium[25], the transfer of the species [Pasteurella]trehalosito the genusBibersteinia[26], the transfer of the species [Pasteurella]ureaeto the genusActinobacillus [27], and the proposed transfer of the species [Haemophilus]ducreyi to a novel genus [28]). However, despite these changes, the classification of the generaActinobacillus, Haemophilus,andPasteurella is still problematic and each genus continues to contain members which exhibit polyphyletic branching [5, 17–20].Multiple studies have attempted to define a core group of species which cluster around the nomenclatural type species ofActinobacillus, Haemophilus,orPasteurella as the only true members of these genera (i.e.,sensu stricto) [13, 15–17, 29–31], but the taxonomy and phylogeny of these bacteria continue to remain inconclusive [20, 32, 33]. Several methods have been employed for the demarcation of these genera; however, no simple method or criterion is available that can clearly delimit these genera. It has been suggested that genome based studies may provide reliable means of clarifying the evolutionary relationships of these bacteria [33].Since the availability of the first complete genome sequence of theHaemophilus influenzae [34], a large number of genomes for the members of the familyPasteurellaceae have become available in public databases [35, 36]. The availability of these genomes provides us with an opportunity to complete comprehensive genome scale phylogenetic analyses of the familyPasteurellaceae. These genome sequences have also been utilized to carry out comparative genomic analyses to identify molecular signatures (namely, conserved signature indels (CSIs) in various proteins), commonly shared by all or closely related subsets of species within the familyPasteurellaceae. On the basis of the molecular signatures identified from comparative analyses ofPasteurellaceae genomes in conjunction with core genome based and multilocus sequence based phylogenetic analyses, we have identifiedsensu stricto clades ofActinobacillus, Haemophilus,andPasteurella that are supported by 3, 7, and 6 unique molecular signatures, respectively. We also report sets of molecular signatures that are unique characteristics of the pathogen containing generaAggregatibacter andMannheimia.


## 2. Methods

### 2.1. Multilocus Sequence Analysis

Multilocus sequence analysis was completed for members of the familyPasteurellaceae using widely available nucleotide sequences of the 16S rDNA,infB (translation initiation factor IF-2),recN (DNA repair protein), andrpoB (DNA-directed RNA polymerase subunit beta) genes which have been used, individually or as part of a set, in a number of previous phylogenetic analyses of the familyPasteurellaceae [15–17, 29, 30]. Gene sequences for these four genes were obtained for 52Pasteurellaceae strains, representing a large majority of the knownPasteurellaceae species, and 2 members ofVibrio cholerae from the NCBI nucleotide database [37]. Species which were missing one of these four genes or which did not have a gene sequence that was at least 50% of the length of the full gene were excluded from the analysis. The four genes were individually aligned using MUSCLE [38] and manually concatenated to create a combined dataset that contained 10 183 nucleotide long alignments. A maximum-likelihood tree based on 100 bootstrap replicates of this alignment was constructed using MEGA 6.0 [39] while employing maximum composite likelihood substitution model.


### 2.2.Pasteurellaceae Core Genome Phylogenetic Tree

A phylogenetic tree of 76Pasteurellaceae strains, rooted using 7 members of the familyVibrionaceae, based on the core genome of the familyPasteurellaceae was created for this study. The core set ofPasteurellaceae proteins were identified using the UCLUST algorithm [55] to identify widely distributed protein families with at least 30% sequence identity and 50% sequence length. Proteins families which were present in less than 50% of the input genomes were excluded from further analysis. Potentially paralogous sequences (additional proteins from the same organism in a single protein family) within the remaining protein families were also excluded from further analysis. Each protein family was individually aligned using MAFFT 7 [56]. Aligned amino acid positions which contained gaps in more than 50% of organisms were excluded from further analysis. The remaining amino acid positions were concatenated to create a combined dataset that contained 128 080 amino acid long alignments. An approximately maximum-likelihood tree based on this alignment was constructed using FastTree 2 [57] while employing the Whelan and Goldman substitution model [58].


### 2.3. Identification of Molecular Signatures (CSIs) for Different Genera of the FamilyPasteurellaceae

The detailed outline of the process of identifying CSIs has been recently published [59]. In brief, Blastp searches were performed on all proteins from the genome ofHaemophilus influenzaeF3047 [47]. Ten to fifteen high scoring homologues that were present inHaemophilus,otherPasteurellaceae, andGammaproteobacteria species were retrieved, and their multiple sequence alignments were constructed using Clustal X 1.83 [60]. The alignments were visually inspected to identify any conserved inserts or deletions (indels) that are restricted to the particular clades of the family Pasteurellaceae, which are flanked on each side by at least 5-6 identical/conserved residues in the neighbouring 30–40 amino acids. The selected sequences containing the indels and their flanking conserved regions were further evaluated by detailed Blastp searches to determine species distribution and group specificity. The results of these Blast searches were processed using Sig_Create and Seq_Style to construct signature files [59]. Due to space constraints, the sequence alignment files presented here contain sequence information for a limited number of species within the orderPasteurellaceaeand a representative selection of outgroup species. However, in each case, all members of the order and outgroups exhibited similar sequence characteristics to the representatives.


## 2.1. Multilocus Sequence Analysis

Multilocus sequence analysis was completed for members of the familyPasteurellaceae using widely available nucleotide sequences of the 16S rDNA,infB (translation initiation factor IF-2),recN (DNA repair protein), andrpoB (DNA-directed RNA polymerase subunit beta) genes which have been used, individually or as part of a set, in a number of previous phylogenetic analyses of the familyPasteurellaceae [15–17, 29, 30]. Gene sequences for these four genes were obtained for 52Pasteurellaceae strains, representing a large majority of the knownPasteurellaceae species, and 2 members ofVibrio cholerae from the NCBI nucleotide database [37]. Species which were missing one of these four genes or which did not have a gene sequence that was at least 50% of the length of the full gene were excluded from the analysis. The four genes were individually aligned using MUSCLE [38] and manually concatenated to create a combined dataset that contained 10 183 nucleotide long alignments. A maximum-likelihood tree based on 100 bootstrap replicates of this alignment was constructed using MEGA 6.0 [39] while employing maximum composite likelihood substitution model.


## 2.2.Pasteurellaceae Core Genome Phylogenetic Tree

A phylogenetic tree of 76Pasteurellaceae strains, rooted using 7 members of the familyVibrionaceae, based on the core genome of the familyPasteurellaceae was created for this study. The core set ofPasteurellaceae proteins were identified using the UCLUST algorithm [55] to identify widely distributed protein families with at least 30% sequence identity and 50% sequence length. Proteins families which were present in less than 50% of the input genomes were excluded from further analysis. Potentially paralogous sequences (additional proteins from the same organism in a single protein family) within the remaining protein families were also excluded from further analysis. Each protein family was individually aligned using MAFFT 7 [56]. Aligned amino acid positions which contained gaps in more than 50% of organisms were excluded from further analysis. The remaining amino acid positions were concatenated to create a combined dataset that contained 128 080 amino acid long alignments. An approximately maximum-likelihood tree based on this alignment was constructed using FastTree 2 [57] while employing the Whelan and Goldman substitution model [58].


## 2.3. Identification of Molecular Signatures (CSIs) for Different Genera of the FamilyPasteurellaceae

The detailed outline of the process of identifying CSIs has been recently published [59]. In brief, Blastp searches were performed on all proteins from the genome ofHaemophilus influenzaeF3047 [47]. Ten to fifteen high scoring homologues that were present inHaemophilus,otherPasteurellaceae, andGammaproteobacteria species were retrieved, and their multiple sequence alignments were constructed using Clustal X 1.83 [60]. The alignments were visually inspected to identify any conserved inserts or deletions (indels) that are restricted to the particular clades of the family Pasteurellaceae, which are flanked on each side by at least 5-6 identical/conserved residues in the neighbouring 30–40 amino acids. The selected sequences containing the indels and their flanking conserved regions were further evaluated by detailed Blastp searches to determine species distribution and group specificity. The results of these Blast searches were processed using Sig_Create and Seq_Style to construct signature files [59]. Due to space constraints, the sequence alignment files presented here contain sequence information for a limited number of species within the orderPasteurellaceaeand a representative selection of outgroup species. However, in each case, all members of the order and outgroups exhibited similar sequence characteristics to the representatives.


## 3. Results and Discussion

### 3.1. Phylogenetic Analysis of thePasteurellaceae

Elucidating an accurate phylogeny of the members of the familyPasteurellaceae has been a long standing challenge inPasteurellaceae research [10–12, 18, 19]. Early 16S rRNA based studies revealed that the established taxonomy of the familyPasteurellaceae was not consistent with their genetically inferred phylogeny [12, 14]. This has led to a long series of taxonomic revisions within the familyPasteurellaceae, a process which is still taking place today [7, 18, 22, 28]. However, it was subsequently discovered that phylogenetic trees ofPasteurellaceae species based on different genes did not completely agree with each other [15, 16, 31]. In particular, phylogenetic trees based on the 16S rRNA gene, often considered the gold standard in bacterial taxonomy and phylogeny [61, 62], disagreed with highly robust multilocus sequence and concatenated protein sequence based phylogenetic trees [9, 17, 19, 20, 53, 63].Phylogenetic trees based on concatenated sequences for a large number of unlinked and conserved loci are more reliable and robust than phylogenetic trees based on any single gene or protein [64, 65]. Due to a rapid increase in the availability of genomic sequence data, we are now able to complete genome scale phylogenetic analyses of the familyPasteurellaceae which cover a vast majority of the diversity within the family. In this work we have produced a phylogenetic tree for 74 genome sequenced members of the familyPasteurellaceae based on 128 080 aligned amino acid positions (Figure 1(a)). The branching patterns of the core genome phylogenetic tree produced in this work largely agree with a previous genome based phylogenetic tree produced for a limited number ofPasteurellaceaespecies [19] and a concatenated protein based phylogenetic tree of the familyPasteurellaceaeproduced by our lab in a previous study [20]. Additionally, we have also produced a multilocus sequence based phylogenetic tree using the 16S rDNA,infB,recN, andrpoB genes which are commonly used in the phylogenetic analysis of the familyPasteurellaceae(Figure 1(b)) [15–17, 29, 30]. This tree also showed broadly similar branching patterns to past multilocus sequence based phylogenetic trees [17, 18] and to our core genome based phylogenetic tree. Both our core genome based and multilocus sequence based phylogenetic 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 similar division of the familyPasteurellaceae into two or more large groups is seen in many other robust multilocus or concatenated protein based phylogenetic trees [17, 19, 53, 63]; however, this division is not readily apparent in phylogenies based on the 16S rRNA gene [9, 66].Figure 1
(a) A maximum-likelihood core genome phylogenetic tree of sequenced members of the familyPasteurellaceae. (b) A maximum-likelihood phylogenetic tree based on concatenated nucleotide sequence alignments of the 16S rDNA,infB,recN, andrpoB genes. Both trees are rooted using members of theVibrionaceae (not shown). Nodes with >80% bootstrap support are indicated by diamond shaped symbols at the node. Clusters of species representingActinobacillus sensu stricto, Haemophilus sensu stricto, Pasteurella sensu stricto,Aggregatibacter, andMannheimia are indicated by brackets. Members of the generaActinobacillus, Haemophilus,andPasteurella which do not fall into their respective “sensu stricto” clades are indicated by the presence of square brackets around their generic name (ex. [Pasteurella]pneumotropica).
(a)
(b)A majority of the known genera within the familyPasteurellaceae form well-defined and coherent clusters in phylogenetic trees (Figure 1) [9, 17, 19, 20, 66]. The generaActinobacillus, Haemophilus,andPasteurella, which were described before the advent of genetic characterization, exhibit polyphyletic branching in all gene and protein based phylogenetic trees, including the core genome based and multilocus sequence based phylogenetic trees created in this work (Figure 1). However, there are large clusters ofActinobacillus, Haemophilus,andPasteurella species identifiable in the phylogenetic trees which represent the core or “sensu stricto” members of each genera. The clusters of species that representActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto are indicated in Figure 1. Members of each genera which fall outside of thesensu stricto clusters, indicated in our phylogenetic trees by the presence of square brackets around their genus name (e.g., [Pasteurella]pneumotropica), are only distantly related to thesensu stricto members of their genus and will require reclassification in order to make their taxonomy and phylogeny concordant.


### 3.2. The Usefulness of Conserved Signature Indels as Phylogenetic and Taxonomic Markers

Whole genome sequences are a rich resource for the discovery of molecular signatures which are unique to a group of organisms [67–69]. One useful class of shared molecular signatures are conserved signature indels (CSIs), which are insertions/deletions uniquely present in protein sequences from a group of evolutionarily related organisms [59, 70, 71]. The unique, shared presence of multiple CSIs by a group of related species is most parsimoniously explained by the occurrence of the genetic changes that resulted in these CSIs in a common ancestor of the group, followed by vertical transmission of these CSIs to various descendant species [59, 71–73]. Hence, these CSIs represent molecular synapomorphies (markers of common evolutionary decent) which can be used to identify and demarcate specific bacterial groups in molecular terms and for understanding their interrelationships independently of phylogenetic trees [59, 70–72]. CSIs have recently been used to propose important taxonomic changes for a number of bacterial groups (namely, Aquificae, Spirochaetes, Thermotogae,Xanthomonadales, andBorrelia) at different taxonomic ranks [69, 74–77]. In the present work, we have completed comprehensive comparative analysis ofPasteurellaceae genomes (Table 1) in order to identify CSIs that are primarily restricted to the different genera within the familyPasteurellaceae. We have identified 3, 7, and 6 unique molecular signatures which are shared byActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto, respectively. Information regarding these CSIs and their evolutionary significances is discussed below.Table 1
Genome characteristics of the sequencedPasteurellaceae included in our analyses.
Organism name
BioProject
Size(Mb)
Proteins
G-C(%)
References
Actinobacillus pleuropneumoniae L20
CP000569
2.27
2013
41.3
[40]
Actinobacillus pleuropneumoniaeserovar 3 str. JL03
CP000687
2.24
2036
41.2
[41]
Actinobacillus pleuropneumoniaeserovar7 str. AP76
CP001091
2.35
2142
41.2
STHHb
Actinobacillus ureaeATCC 25976a
AEVG0
2.30
2475
—
BCMg
Actinobacillus minor202a
ACFT0
2.13
2050
39.3
McGill University
Actinobacillus minorNM305a
ACQL0
2.43
2411
39.3
McGill University
Actinobacillus succinogenes130Z
CP000746
2.32
2079
44.9
Joint Genome Institute
Aggregatibacter aphrophilusNJ8700
CP001607
2.31
2219
42.2
[19, 42]
Aggregatibacter actinomycetemcomitansD11S-1
CP001733
2.20
2280
44.3
[43]
Aggregatibacter actinomycetemcomitansD7S-1
CP003496
2.31
2250
44.3
[44]
Aggregatibacter segnisATCC 33393a
AEPS0
1.99
1956
—
BCMg
Gallibacterium anatisUMN179
CP002667
2.69
2500
39.9
[45]
Haemophilus aegyptiusATCC 11116a
AFBC0
1.92
2020
—
BCMg
Haemophilus ducreyi35000HP
AE017143
1.70
1717
38.2
Ohio State University
Haemophilus haemolyticusM21621a
AFQQ0
2.09
1894
—
[46]
Haemophilus influenzae10810
FQ312006
1.98
1903
38.1
WTSIh
Haemophilus influenzaeF3031
FQ670178
1.99
1770
38.2
[47]
Haemophilus influenzaeF3047
FQ670204
2.01
1786
38.2
[47]
Haemophilus influenzae22.1-21a
AAZD0
1.89
2224
38.0
[48]
Haemophilus influenzae3655
AAZF0
1.88
1929
38.0
[48]
Haemophilus influenzae6P18H1a
ABWW0
1.91
1893
38.2
CGS, ASRIe
Haemophilus influenzae7P49H1a
ABWV0
1.83
1752
37.9
CGS, ASRIe
Haemophilus influenzaeNT127a
ACSL0
1.87
1809
38.0
BIGSPc
Haemophilus influenzaePittAAa
AAZG0
1.88
1981
38.1
[48]
Haemophilus influenzaePittIIa
AAZI0
1.95
2028
38.0
[48]
Haemophilus influenzaePittHHa
AAZH0
1.84
1977
38.0
[48]
Haemophilus influenzaeR3021a
AAZJ0
1.88
2307
37.9
[48]
Haemophilus influenzaeRdAWa
ACSM0
1.80
1718
38.0
BIGSPc
Haemophilus influenzae86-028NP
CP000057
1.91
1792
38.2
[49]
Haemophilus influenzaePittEE
CP000671
1.81
1613
38.0
[48]
Haemophilus influenzaePittGG
CP000672
1.89
1661
38.0
[48]
Haemophilus influenzaeRd KW20
L42023
1.83
1657
38.2
[34]
Haemophilus influenzaeR2846
CP002276
1.82
1636
38.0
UW-SBRId
Haemophilus influenzaeR2866
CP002277
1.93
1795
38.1
UW-SBRId
Haemophilus parainfluenzaeATCC 33392a
AEWU0
2.11
2010
—
BCMg
Haemophilus parainfluenzaeT3T1
FQ312002
2.09
1975
39.6
WTSIh
Haemophilus parasuis29755a
ABKM0
2.22
2244
39.8
Iowa State University
Haemophilus parasuisSH0165
CP001321
2.27
2021
40.0
[50]
Haemophilus pittmaniaeHK 85a
AFUV0
2.18
2390
—
J. Craig Venter Institute
Haemophilus sputorumCCUG13788a
AFNK0
2.14
2073
—
Aarhus University Hospital
Haemophilus parahaemolyticusHK385a
AJSW0
1.81
1764
—
J. Craig Venter Institute
Haemophilus paraphrohaemolyticusHK411a
AJMU0
2.02
2025
—
J. Craig Venter Institute
Haemophilus sp. oral taxon 851str.F0397a
AGRK0
1.84
1809
—
GCG-WUf
Histophilus somni2336
CP000947
2.26
1980
37.4
Joint Genome Institute
Histophilus somni129PT
CP000436
2.01
1798
37.2
[51]
Mannheimia succiniciproducensMBEL55E
AE016827
2.31
2370
42.5
[52]
Mannheimia haemolytica PHL213a
AASA0
2.57
2695
41.1
[53]
Pasteurella multocidasubsp. multocidastr. Pm70
AE004439
2.26
2012
40.4
[54]
Pasteurella dagmatisATCC 43325a
ACZR0
2.25
2053
37.4
BCMg
aThe genomes of these species/strains are currently under scaffolds/contigs status.
bStiftung 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 Medicine.
gBaylor College of Medicine (BCM).
hWellcome Trust Sanger Institute (WTSI).


### 3.3. Molecular Signatures Specific forActinobacillus sensu stricto

The genusActinobacillus was originally defined as a group of growth factor independent host-associated rods which shared phenotypic or biochemical similarity withActinobacillus lignieresii, the type species of the genus [24, 78]. However, the original classification scheme for the genusActinobacillusled to the inclusion of a highly heterogeneous and polyphyletic grouping of species within the genus [12–14]. An assemblage ofActinobacillus species closely related toActinobacillus lignieresii has been recognized asActinobacillus sensu stricto (i.e., the core members of the genusActinobacillus) in both our phylogenetic analysis (Figure 1) and past phylogenetic analyses [12–14, 17]. Differentiation ofActinobacillus sensu stricto from otherActinobacillus species and the modern criteria for placing novel species within the genusActinobacillus sensu stricto is heavily reliant on genetic and genomic criteria, namely, DNA-DNA hybridization values, 16S rRNA sequence similarity, and other single gene sequence comparisons [13, 18]. There are currently no known discrete characteristics which are unique toActinobacillus that define the genus. In this work, we have completed a comprehensive comparative analysis ofPasteurellaceae genomes in order to identify unique, defining molecular signatures for different genera within the familyPasteurellaceae. We have identified 3 CSIs which are unique, defining molecular signatures for the sequenced members ofActinobacillus sensu stricto(namely,Actinobacillus capsulatus, A. pleuropneumoniae, A. suis,andA. ureae). An example of a CSI specific forActinobacillus sensu stricto is shown in Figure 2. The CSI consists of a 1-amino-acid insertion in a conserved region of a 3′-nucleotidase which is present in all sequenced members ofActinobacillus sensu stricto and absent in all other sequencedGammaproteobacteria. Sequence information for 2 other CSIs which are also unique characteristics of theActinobacillus sensu stricto clade is presented in Supplemental Figures 1
-
2 available online at http://dx.doi.org/10.1155/2015/198560 and their characteristics are briefly summarized in Table 2
(
A
).Table 2
Conserved signature indels specific for genera within the familyPasteurellaceae.
Protein name
Gene name
GenBank identifier
Figure number
Indel size
Indel positiona
(A) CSIs specific forActinobacillus  sensu stricto
3′-nucleotidase
surE
126208128
Figure2
1 aa ins
367–402
GTP pyrophosphokinase
relA
126207889
Sup. Figure1
1 aa ins
368–412
Anaerobic glycerol-3-phosphate dehydrogenase subunit
glpA
491834528
Sup. Figure2
1 aa ins
359–400
(B) CSIs specific forHaemophilus  sensu stricto
Biotin-protein ligase
birA
144979005
Figure3
6 aa del
138–178
Aspartate ammonia-lyase
aspA
145630289
Sup. Figure3
1 aa ins
34–75
NAD(P) transhydrogenase subunit alpha
pntA
145631394
Sup.  Figure4
1 aa del
352–378
Fumarate reductase subunit C
frdC
301169552
Sup.  Figure5
3 aa ins
31–89
Hypothetical tRNA/rRNA methyltransferase
—
145636352
Sup.  Figure6
1 aa del
17–58
Gamma-glutamyl kinase
proB
145629980
Sup.  Figure7
1 aa ins
197–253
ACP phosphodiesterase
acpD
68250119
Sup.  Figure8
2 aa del
119–159
(C) CSIs specific forPasteurella sensu stricto
Menaquinone-specific isochorismate synthase
menF
386834899
Figure4
4 aa ins
29–86
tRNA s(4)U8 sulfurtransferase
thiI
15602400
Sup.  Figure9
2 aa del
412–446
FKBP-type peptidyl-prolyl cis-trans isomerase
slyD
378775595
Sup.  Figure10
2 aa del
151–188
Aspartate-semialdehyde dehydrogenase
asd
383311492
Sup.  Figure11
1 aa del
173–245
Lactate permease family transporter
lldP
492154065
Sup.  Figure12
2 aa ins
390–427
Cell division proteinftsA
ftsA
492155843
Sup.  Figure13
1 aa ins
357–387
(D) CSIs specific forAggregatibacter
nhaC family sodium:proton antiporter
nhaC
493769836
Figure5(a)
3 aa ins
396–437
Outer membrane protein
omp
261866907
Sup.  Figure14
4 aa del
25–64
Multidrug transportermurJ
murJ
365966332
Sup.  Figure15
1 aa del
190–220
NADH dehydrogenase
nuoE
387120244
Sup.  Figure16
1 aa ins
372–412
(E)  CSIs specific forMannheimia
Methyl-galactoside ABC transporter substrate-binding protein
—
472335016
Figure5(b)
1 aa del
33–73
UDP-N-acetylmuramoylalanyl-D-glutamate–2,6-diaminopimelate ligase
murE
472333011
Sup.  Figure17
2 aa del
418–473
Glutathione-regulated potassium-efflux protein
kefC
472333189
Sup.  Figure18
1 aa ins
504–531
Glycerol-3-phosphate acyltransferase
plsB
472334521
Sup.  Figure19
2 aa del
214–252Figure 2
A partial sequence alignment of a 3′-nucleotidase showing a 1-amino-acid insertion identified in all members ofActinobacillus sensu stricto. This insertion was not found in the homologues from any member of the genusActinobacillusthat was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for a representative subset of the familyPasteurellaceae and the classGammaproteobacteria is shown, but unless otherwise indicated, similar CSIs were detected in all members of the indicated group and not detected in any other bacterial species in the top 250 BLAST hits. The dashes (-) in the alignments indicate identity with the residue in the top sequence. GenBank identification (GI) numbers for each sequence are indicated in the second column. Sequence information for other CSIs specific toActinobacillus sensu stricto are presented in Supplemental Figures  1-2 and their characteristics are summarized in Table 2
(
A
).


### 3.4. Molecular Signatures Specific forHaemophilus sensu stricto

The classification of novel species into the genusHaemophilus was initially based on phenotypic and biochemical properties, most importantly, the dependence of growth on the presence of factor V and factor X in blood [13, 78, 79]. As withActinobacillus, the classification ofHaemophilus on the basis of phenotypic and biochemical properties has led to the genus containing an extremely heterogeneous group of species [12–14, 32]. Species from the genusHaemophilus have undergone a number of transfers and reclassifications [21–24, 28]. However, the genus remains highly polyphyletic (Figure 1) [17, 19, 28]. The core members of the genusHaemophilus (namely,Haemophilus sensu stricto) consist ofHaemophilus influenzae, H. aegyptius, andH. haemolyticus based on 16S rRNA sequence analysis [12–14, 32]. However, phylogenetic analysis based on DNA-DNA hybridization and multilocus sequence analysis suggests thatH. parainfluenzaeandH. pittmaniae are also members ofHaemophilus sensu stricto [30, 80]. Phylogenetic analysis ofrpoB,infB, and concatenated gene sets also suggest that [Pasteurella]pneumotropicaand related isolates are closely related toHaemophilus sensu stricto [15, 16].Our comparative analysis ofPasteurellaceae genomes has led to the identification of 7 CSIs that are unique characteristics ofHaemophilus sensu stricto which consists ofHaemophilus influenzae, H. aegyptius, H. haemolyticus, H. parainfluenzae, H. pittmaniae, and [Pasteurella]pneumotropica(Figure 1). One example of a CSI specific for the members ofHaemophilus sensu stricto, shown in Figure 3, consists of a 4-amino-acid deletion in a biotin-protein ligase which is uniquely found in homologs fromHaemophilus sensu stricto and absent in all other sequencedGammaproteobacteria. Sequence information for 6 additional CSIs which are also unique characteristics ofHaemophilus sensu stricto is presented in Supplemental Figures  3–8 and their characteristics are briefly summarized in Table 2
(
B
). These CSIs and our phylogenetic trees (Figure 1) suggest thatHaemophilus influenzae, H. aegyptius, H. haemolyticus, H. parainfluenzae, H. pittmaniae, and [Pasteurella]pneumotropicashare a close evolutionary relationship and should all be considered members ofHaemophilus sensu stricto. Additionally, these results also suggest that [Pasteurella]pneumotropica is incorrectly classified as a member of the genusPasteurella and should be reclassified as “Haemophilus pneumotropica.”Figure 3
A partial sequence alignment of 1,4-dihydroxy-2-naphthoate octaprenyltransferase showing a 2-amino-acid insertion identified in all members ofHaemophilus sensu stricto. This insertion was not found in the homologues from any member of the genusHaemophilusthat was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for other CSIs specific toHaemophilus sensu stricto is presented in Supplemental Figures  3–8 and their characteristics are summarized in Table 2
(
B
).


### 3.5. Molecular Signatures Specific forPasteurella sensu stricto

The genusPasteurella is highly heterogeneous and polyphyletic (Figure 1) [13]. Similar to the members ofActinobacillus, bacterial isolates were originally classified as members of the genusPasteurella based on growth factor independent growth and phenotypic or biochemical similarity toPasteurella multocida, the type species of the genus [78, 81]. The monophyletic clusters ofPasteurellaspecies that branch withPasteurella multocida are considered the core members of the genus (namely,Pasteurella sensu stricto) [9, 13, 16, 17]. Our comparative analysis ofPasteurellaceae genomes has led to the identification of 6 CSIs which are unique characteristics for the sequenced members ofPasteurella sensu stricto(namely,Pasteurella multocidaandP. dagmatis). An example of a CSI uniquely found in the sequenced members ofPasteurella sensu stricto, consisting of a 4-amino-acid insertion in a conserved region of Menaquinone-specific isochorismate synthase, is shown in Figure 4. This CSI is only found in the sequenced members ofPasteurella sensu stricto and is absent from all other sequencedGammaproteobacteria. Partial sequence alignments for 5 additional CSIs which are also unique characteristics ofPasteurella sensu stricto are presented in Supplemental Figures 9
–
13 and their characteristics are briefly summarized in Table 2
(
C
).Figure 4
A partial sequence alignment of Menaquinone-specific isochorismate synthase showing a 4-amino-acid insertion identified in all members ofPasteurella sensu stricto. This insertion was not found in the homologues from any member of the genusPasteurella that was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for other CSIs specific toPasteurella sensu stricto is presented in Supplemental Figures  9–13 and their characteristics are summarized in Table 2
(
C
).


### 3.6. Molecular Signatures Specific for the GeneraAggregatibacter orMannheimia

The genusAggregatibacter was proposed as a novel taxonomic classification for a monophyletic cluster ofActinobacillusandHaemophilus species which branched distinctly from the “sensu stricto” members of their respective clades [21]. Similarly, the genusMannheimia was proposed as a novel classification for thePasteurella Haemolytica complex which did not branch withPasteurella sensu stricto in phylogenetic trees [7]. Currently other than branching in phylogenetic trees or relatedness in DNA-DNA hybridization studies, the members of the generaAggregatibacter orMannheimia do not share any single unique or defining biochemical or molecular characteristic that can differentiate them from all other bacteria [5, 82].In this study we have identified 4 CSIs that are unique molecular characteristics shared by all sequenced species of the genusAggregatibacter and another 4 CSIs which are uniquely found in all sequenced members of the genusMannheimia. Examples of CSIs specific to the sequenced members ofAggregatibacter andMannheimia are shown in Figure 5. A partial sequence alignment of anhaC family sodium:proton antiporter containing a 3-amino-acid insertion specific for all sequenced species of the genusAggregatibacter is shown in Figure 5(a) and a partial sequence alignment of a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specific for all sequenced species of the genusMannheimia is shown in Figure 5(b). In each case, the identified CSIs were only found in the sequenced members of the generaAggregatibacter orMannheimia and were absent from all other sequencedGammaproteobacteria. Partial sequence alignments additional CSIs specific for the generaAggregatibacter orMannheimia are provided in Supplemental Figures 14
–
19 and their characteristics are summarized in Tables 2
(
D
)
-
2
(
E
). These CSIs are the first discrete molecular characteristics which are unique for the generaAggregatibacter andMannheimia and support their observed monophyly in phylogenetic trees. Additionally, these CSIs could be useful targets for the development of PCR based diagnostic assays for the generaAggregatibacter andMannheimia which amplify the CSI containing DNA segment using the conserved flanking regions of the CSIs [83, 84].Figure 5
A partial sequence alignment of (a) anhaC family sodium:proton antiporter containing a 3-amino-acid insertion specific for all sequenced species of the genusAggregatibacter (b) a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specific for all sequenced species of the genusMannheimia. In each case, the identified CSIs were only found in the sequenced members of the generaAggregatibacter orMannheimia and were absent from all other sequencedGammaproteobacteria. Sequence information for other CSIs specific toAggregatibacter orMannheimiais presented in Supplemental Figures  14–19 and their characteristics are summarized in Tables 2
(
D
) and 2
(
E
).
(a)
(b)


## 3.1. Phylogenetic Analysis of thePasteurellaceae

Elucidating an accurate phylogeny of the members of the familyPasteurellaceae has been a long standing challenge inPasteurellaceae research [10–12, 18, 19]. Early 16S rRNA based studies revealed that the established taxonomy of the familyPasteurellaceae was not consistent with their genetically inferred phylogeny [12, 14]. This has led to a long series of taxonomic revisions within the familyPasteurellaceae, a process which is still taking place today [7, 18, 22, 28]. However, it was subsequently discovered that phylogenetic trees ofPasteurellaceae species based on different genes did not completely agree with each other [15, 16, 31]. In particular, phylogenetic trees based on the 16S rRNA gene, often considered the gold standard in bacterial taxonomy and phylogeny [61, 62], disagreed with highly robust multilocus sequence and concatenated protein sequence based phylogenetic trees [9, 17, 19, 20, 53, 63].Phylogenetic trees based on concatenated sequences for a large number of unlinked and conserved loci are more reliable and robust than phylogenetic trees based on any single gene or protein [64, 65]. Due to a rapid increase in the availability of genomic sequence data, we are now able to complete genome scale phylogenetic analyses of the familyPasteurellaceae which cover a vast majority of the diversity within the family. In this work we have produced a phylogenetic tree for 74 genome sequenced members of the familyPasteurellaceae based on 128 080 aligned amino acid positions (Figure 1(a)). The branching patterns of the core genome phylogenetic tree produced in this work largely agree with a previous genome based phylogenetic tree produced for a limited number ofPasteurellaceaespecies [19] and a concatenated protein based phylogenetic tree of the familyPasteurellaceaeproduced by our lab in a previous study [20]. Additionally, we have also produced a multilocus sequence based phylogenetic tree using the 16S rDNA,infB,recN, andrpoB genes which are commonly used in the phylogenetic analysis of the familyPasteurellaceae(Figure 1(b)) [15–17, 29, 30]. This tree also showed broadly similar branching patterns to past multilocus sequence based phylogenetic trees [17, 18] and to our core genome based phylogenetic tree. Both our core genome based and multilocus sequence based phylogenetic 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 similar division of the familyPasteurellaceae into two or more large groups is seen in many other robust multilocus or concatenated protein based phylogenetic trees [17, 19, 53, 63]; however, this division is not readily apparent in phylogenies based on the 16S rRNA gene [9, 66].Figure 1
(a) A maximum-likelihood core genome phylogenetic tree of sequenced members of the familyPasteurellaceae. (b) A maximum-likelihood phylogenetic tree based on concatenated nucleotide sequence alignments of the 16S rDNA,infB,recN, andrpoB genes. Both trees are rooted using members of theVibrionaceae (not shown). Nodes with >80% bootstrap support are indicated by diamond shaped symbols at the node. Clusters of species representingActinobacillus sensu stricto, Haemophilus sensu stricto, Pasteurella sensu stricto,Aggregatibacter, andMannheimia are indicated by brackets. Members of the generaActinobacillus, Haemophilus,andPasteurella which do not fall into their respective “sensu stricto” clades are indicated by the presence of square brackets around their generic name (ex. [Pasteurella]pneumotropica).
(a)
(b)A majority of the known genera within the familyPasteurellaceae form well-defined and coherent clusters in phylogenetic trees (Figure 1) [9, 17, 19, 20, 66]. The generaActinobacillus, Haemophilus,andPasteurella, which were described before the advent of genetic characterization, exhibit polyphyletic branching in all gene and protein based phylogenetic trees, including the core genome based and multilocus sequence based phylogenetic trees created in this work (Figure 1). However, there are large clusters ofActinobacillus, Haemophilus,andPasteurella species identifiable in the phylogenetic trees which represent the core or “sensu stricto” members of each genera. The clusters of species that representActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto are indicated in Figure 1. Members of each genera which fall outside of thesensu stricto clusters, indicated in our phylogenetic trees by the presence of square brackets around their genus name (e.g., [Pasteurella]pneumotropica), are only distantly related to thesensu stricto members of their genus and will require reclassification in order to make their taxonomy and phylogeny concordant.


## 3.2. The Usefulness of Conserved Signature Indels as Phylogenetic and Taxonomic Markers

Whole genome sequences are a rich resource for the discovery of molecular signatures which are unique to a group of organisms [67–69]. One useful class of shared molecular signatures are conserved signature indels (CSIs), which are insertions/deletions uniquely present in protein sequences from a group of evolutionarily related organisms [59, 70, 71]. The unique, shared presence of multiple CSIs by a group of related species is most parsimoniously explained by the occurrence of the genetic changes that resulted in these CSIs in a common ancestor of the group, followed by vertical transmission of these CSIs to various descendant species [59, 71–73]. Hence, these CSIs represent molecular synapomorphies (markers of common evolutionary decent) which can be used to identify and demarcate specific bacterial groups in molecular terms and for understanding their interrelationships independently of phylogenetic trees [59, 70–72]. CSIs have recently been used to propose important taxonomic changes for a number of bacterial groups (namely, Aquificae, Spirochaetes, Thermotogae,Xanthomonadales, andBorrelia) at different taxonomic ranks [69, 74–77]. In the present work, we have completed comprehensive comparative analysis ofPasteurellaceae genomes (Table 1) in order to identify CSIs that are primarily restricted to the different genera within the familyPasteurellaceae. We have identified 3, 7, and 6 unique molecular signatures which are shared byActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto, respectively. Information regarding these CSIs and their evolutionary significances is discussed below.Table 1
Genome characteristics of the sequencedPasteurellaceae included in our analyses.
Organism name
BioProject
Size(Mb)
Proteins
G-C(%)
References
Actinobacillus pleuropneumoniae L20
CP000569
2.27
2013
41.3
[40]
Actinobacillus pleuropneumoniaeserovar 3 str. JL03
CP000687
2.24
2036
41.2
[41]
Actinobacillus pleuropneumoniaeserovar7 str. AP76
CP001091
2.35
2142
41.2
STHHb
Actinobacillus ureaeATCC 25976a
AEVG0
2.30
2475
—
BCMg
Actinobacillus minor202a
ACFT0
2.13
2050
39.3
McGill University
Actinobacillus minorNM305a
ACQL0
2.43
2411
39.3
McGill University
Actinobacillus succinogenes130Z
CP000746
2.32
2079
44.9
Joint Genome Institute
Aggregatibacter aphrophilusNJ8700
CP001607
2.31
2219
42.2
[19, 42]
Aggregatibacter actinomycetemcomitansD11S-1
CP001733
2.20
2280
44.3
[43]
Aggregatibacter actinomycetemcomitansD7S-1
CP003496
2.31
2250
44.3
[44]
Aggregatibacter segnisATCC 33393a
AEPS0
1.99
1956
—
BCMg
Gallibacterium anatisUMN179
CP002667
2.69
2500
39.9
[45]
Haemophilus aegyptiusATCC 11116a
AFBC0
1.92
2020
—
BCMg
Haemophilus ducreyi35000HP
AE017143
1.70
1717
38.2
Ohio State University
Haemophilus haemolyticusM21621a
AFQQ0
2.09
1894
—
[46]
Haemophilus influenzae10810
FQ312006
1.98
1903
38.1
WTSIh
Haemophilus influenzaeF3031
FQ670178
1.99
1770
38.2
[47]
Haemophilus influenzaeF3047
FQ670204
2.01
1786
38.2
[47]
Haemophilus influenzae22.1-21a
AAZD0
1.89
2224
38.0
[48]
Haemophilus influenzae3655
AAZF0
1.88
1929
38.0
[48]
Haemophilus influenzae6P18H1a
ABWW0
1.91
1893
38.2
CGS, ASRIe
Haemophilus influenzae7P49H1a
ABWV0
1.83
1752
37.9
CGS, ASRIe
Haemophilus influenzaeNT127a
ACSL0
1.87
1809
38.0
BIGSPc
Haemophilus influenzaePittAAa
AAZG0
1.88
1981
38.1
[48]
Haemophilus influenzaePittIIa
AAZI0
1.95
2028
38.0
[48]
Haemophilus influenzaePittHHa
AAZH0
1.84
1977
38.0
[48]
Haemophilus influenzaeR3021a
AAZJ0
1.88
2307
37.9
[48]
Haemophilus influenzaeRdAWa
ACSM0
1.80
1718
38.0
BIGSPc
Haemophilus influenzae86-028NP
CP000057
1.91
1792
38.2
[49]
Haemophilus influenzaePittEE
CP000671
1.81
1613
38.0
[48]
Haemophilus influenzaePittGG
CP000672
1.89
1661
38.0
[48]
Haemophilus influenzaeRd KW20
L42023
1.83
1657
38.2
[34]
Haemophilus influenzaeR2846
CP002276
1.82
1636
38.0
UW-SBRId
Haemophilus influenzaeR2866
CP002277
1.93
1795
38.1
UW-SBRId
Haemophilus parainfluenzaeATCC 33392a
AEWU0
2.11
2010
—
BCMg
Haemophilus parainfluenzaeT3T1
FQ312002
2.09
1975
39.6
WTSIh
Haemophilus parasuis29755a
ABKM0
2.22
2244
39.8
Iowa State University
Haemophilus parasuisSH0165
CP001321
2.27
2021
40.0
[50]
Haemophilus pittmaniaeHK 85a
AFUV0
2.18
2390
—
J. Craig Venter Institute
Haemophilus sputorumCCUG13788a
AFNK0
2.14
2073
—
Aarhus University Hospital
Haemophilus parahaemolyticusHK385a
AJSW0
1.81
1764
—
J. Craig Venter Institute
Haemophilus paraphrohaemolyticusHK411a
AJMU0
2.02
2025
—
J. Craig Venter Institute
Haemophilus sp. oral taxon 851str.F0397a
AGRK0
1.84
1809
—
GCG-WUf
Histophilus somni2336
CP000947
2.26
1980
37.4
Joint Genome Institute
Histophilus somni129PT
CP000436
2.01
1798
37.2
[51]
Mannheimia succiniciproducensMBEL55E
AE016827
2.31
2370
42.5
[52]
Mannheimia haemolytica PHL213a
AASA0
2.57
2695
41.1
[53]
Pasteurella multocidasubsp. multocidastr. Pm70
AE004439
2.26
2012
40.4
[54]
Pasteurella dagmatisATCC 43325a
ACZR0
2.25
2053
37.4
BCMg
aThe genomes of these species/strains are currently under scaffolds/contigs status.
bStiftung 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 Medicine.
gBaylor College of Medicine (BCM).
hWellcome Trust Sanger Institute (WTSI).


## 3.3. Molecular Signatures Specific forActinobacillus sensu stricto

The genusActinobacillus was originally defined as a group of growth factor independent host-associated rods which shared phenotypic or biochemical similarity withActinobacillus lignieresii, the type species of the genus [24, 78]. However, the original classification scheme for the genusActinobacillusled to the inclusion of a highly heterogeneous and polyphyletic grouping of species within the genus [12–14]. An assemblage ofActinobacillus species closely related toActinobacillus lignieresii has been recognized asActinobacillus sensu stricto (i.e., the core members of the genusActinobacillus) in both our phylogenetic analysis (Figure 1) and past phylogenetic analyses [12–14, 17]. Differentiation ofActinobacillus sensu stricto from otherActinobacillus species and the modern criteria for placing novel species within the genusActinobacillus sensu stricto is heavily reliant on genetic and genomic criteria, namely, DNA-DNA hybridization values, 16S rRNA sequence similarity, and other single gene sequence comparisons [13, 18]. There are currently no known discrete characteristics which are unique toActinobacillus that define the genus. In this work, we have completed a comprehensive comparative analysis ofPasteurellaceae genomes in order to identify unique, defining molecular signatures for different genera within the familyPasteurellaceae. We have identified 3 CSIs which are unique, defining molecular signatures for the sequenced members ofActinobacillus sensu stricto(namely,Actinobacillus capsulatus, A. pleuropneumoniae, A. suis,andA. ureae). An example of a CSI specific forActinobacillus sensu stricto is shown in Figure 2. The CSI consists of a 1-amino-acid insertion in a conserved region of a 3′-nucleotidase which is present in all sequenced members ofActinobacillus sensu stricto and absent in all other sequencedGammaproteobacteria. Sequence information for 2 other CSIs which are also unique characteristics of theActinobacillus sensu stricto clade is presented in Supplemental Figures 1
-
2 available online at http://dx.doi.org/10.1155/2015/198560 and their characteristics are briefly summarized in Table 2
(
A
).Table 2
Conserved signature indels specific for genera within the familyPasteurellaceae.
Protein name
Gene name
GenBank identifier
Figure number
Indel size
Indel positiona
(A) CSIs specific forActinobacillus  sensu stricto
3′-nucleotidase
surE
126208128
Figure2
1 aa ins
367–402
GTP pyrophosphokinase
relA
126207889
Sup. Figure1
1 aa ins
368–412
Anaerobic glycerol-3-phosphate dehydrogenase subunit
glpA
491834528
Sup. Figure2
1 aa ins
359–400
(B) CSIs specific forHaemophilus  sensu stricto
Biotin-protein ligase
birA
144979005
Figure3
6 aa del
138–178
Aspartate ammonia-lyase
aspA
145630289
Sup. Figure3
1 aa ins
34–75
NAD(P) transhydrogenase subunit alpha
pntA
145631394
Sup.  Figure4
1 aa del
352–378
Fumarate reductase subunit C
frdC
301169552
Sup.  Figure5
3 aa ins
31–89
Hypothetical tRNA/rRNA methyltransferase
—
145636352
Sup.  Figure6
1 aa del
17–58
Gamma-glutamyl kinase
proB
145629980
Sup.  Figure7
1 aa ins
197–253
ACP phosphodiesterase
acpD
68250119
Sup.  Figure8
2 aa del
119–159
(C) CSIs specific forPasteurella sensu stricto
Menaquinone-specific isochorismate synthase
menF
386834899
Figure4
4 aa ins
29–86
tRNA s(4)U8 sulfurtransferase
thiI
15602400
Sup.  Figure9
2 aa del
412–446
FKBP-type peptidyl-prolyl cis-trans isomerase
slyD
378775595
Sup.  Figure10
2 aa del
151–188
Aspartate-semialdehyde dehydrogenase
asd
383311492
Sup.  Figure11
1 aa del
173–245
Lactate permease family transporter
lldP
492154065
Sup.  Figure12
2 aa ins
390–427
Cell division proteinftsA
ftsA
492155843
Sup.  Figure13
1 aa ins
357–387
(D) CSIs specific forAggregatibacter
nhaC family sodium:proton antiporter
nhaC
493769836
Figure5(a)
3 aa ins
396–437
Outer membrane protein
omp
261866907
Sup.  Figure14
4 aa del
25–64
Multidrug transportermurJ
murJ
365966332
Sup.  Figure15
1 aa del
190–220
NADH dehydrogenase
nuoE
387120244
Sup.  Figure16
1 aa ins
372–412
(E)  CSIs specific forMannheimia
Methyl-galactoside ABC transporter substrate-binding protein
—
472335016
Figure5(b)
1 aa del
33–73
UDP-N-acetylmuramoylalanyl-D-glutamate–2,6-diaminopimelate ligase
murE
472333011
Sup.  Figure17
2 aa del
418–473
Glutathione-regulated potassium-efflux protein
kefC
472333189
Sup.  Figure18
1 aa ins
504–531
Glycerol-3-phosphate acyltransferase
plsB
472334521
Sup.  Figure19
2 aa del
214–252Figure 2
A partial sequence alignment of a 3′-nucleotidase showing a 1-amino-acid insertion identified in all members ofActinobacillus sensu stricto. This insertion was not found in the homologues from any member of the genusActinobacillusthat was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for a representative subset of the familyPasteurellaceae and the classGammaproteobacteria is shown, but unless otherwise indicated, similar CSIs were detected in all members of the indicated group and not detected in any other bacterial species in the top 250 BLAST hits. The dashes (-) in the alignments indicate identity with the residue in the top sequence. GenBank identification (GI) numbers for each sequence are indicated in the second column. Sequence information for other CSIs specific toActinobacillus sensu stricto are presented in Supplemental Figures  1-2 and their characteristics are summarized in Table 2
(
A
).


## 3.4. Molecular Signatures Specific forHaemophilus sensu stricto

The classification of novel species into the genusHaemophilus was initially based on phenotypic and biochemical properties, most importantly, the dependence of growth on the presence of factor V and factor X in blood [13, 78, 79]. As withActinobacillus, the classification ofHaemophilus on the basis of phenotypic and biochemical properties has led to the genus containing an extremely heterogeneous group of species [12–14, 32]. Species from the genusHaemophilus have undergone a number of transfers and reclassifications [21–24, 28]. However, the genus remains highly polyphyletic (Figure 1) [17, 19, 28]. The core members of the genusHaemophilus (namely,Haemophilus sensu stricto) consist ofHaemophilus influenzae, H. aegyptius, andH. haemolyticus based on 16S rRNA sequence analysis [12–14, 32]. However, phylogenetic analysis based on DNA-DNA hybridization and multilocus sequence analysis suggests thatH. parainfluenzaeandH. pittmaniae are also members ofHaemophilus sensu stricto [30, 80]. Phylogenetic analysis ofrpoB,infB, and concatenated gene sets also suggest that [Pasteurella]pneumotropicaand related isolates are closely related toHaemophilus sensu stricto [15, 16].Our comparative analysis ofPasteurellaceae genomes has led to the identification of 7 CSIs that are unique characteristics ofHaemophilus sensu stricto which consists ofHaemophilus influenzae, H. aegyptius, H. haemolyticus, H. parainfluenzae, H. pittmaniae, and [Pasteurella]pneumotropica(Figure 1). One example of a CSI specific for the members ofHaemophilus sensu stricto, shown in Figure 3, consists of a 4-amino-acid deletion in a biotin-protein ligase which is uniquely found in homologs fromHaemophilus sensu stricto and absent in all other sequencedGammaproteobacteria. Sequence information for 6 additional CSIs which are also unique characteristics ofHaemophilus sensu stricto is presented in Supplemental Figures  3–8 and their characteristics are briefly summarized in Table 2
(
B
). These CSIs and our phylogenetic trees (Figure 1) suggest thatHaemophilus influenzae, H. aegyptius, H. haemolyticus, H. parainfluenzae, H. pittmaniae, and [Pasteurella]pneumotropicashare a close evolutionary relationship and should all be considered members ofHaemophilus sensu stricto. Additionally, these results also suggest that [Pasteurella]pneumotropica is incorrectly classified as a member of the genusPasteurella and should be reclassified as “Haemophilus pneumotropica.”Figure 3
A partial sequence alignment of 1,4-dihydroxy-2-naphthoate octaprenyltransferase showing a 2-amino-acid insertion identified in all members ofHaemophilus sensu stricto. This insertion was not found in the homologues from any member of the genusHaemophilusthat was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for other CSIs specific toHaemophilus sensu stricto is presented in Supplemental Figures  3–8 and their characteristics are summarized in Table 2
(
B
).


## 3.5. Molecular Signatures Specific forPasteurella sensu stricto

The genusPasteurella is highly heterogeneous and polyphyletic (Figure 1) [13]. Similar to the members ofActinobacillus, bacterial isolates were originally classified as members of the genusPasteurella based on growth factor independent growth and phenotypic or biochemical similarity toPasteurella multocida, the type species of the genus [78, 81]. The monophyletic clusters ofPasteurellaspecies that branch withPasteurella multocida are considered the core members of the genus (namely,Pasteurella sensu stricto) [9, 13, 16, 17]. Our comparative analysis ofPasteurellaceae genomes has led to the identification of 6 CSIs which are unique characteristics for the sequenced members ofPasteurella sensu stricto(namely,Pasteurella multocidaandP. dagmatis). An example of a CSI uniquely found in the sequenced members ofPasteurella sensu stricto, consisting of a 4-amino-acid insertion in a conserved region of Menaquinone-specific isochorismate synthase, is shown in Figure 4. This CSI is only found in the sequenced members ofPasteurella sensu stricto and is absent from all other sequencedGammaproteobacteria. Partial sequence alignments for 5 additional CSIs which are also unique characteristics ofPasteurella sensu stricto are presented in Supplemental Figures 9
–
13 and their characteristics are briefly summarized in Table 2
(
C
).Figure 4
A partial sequence alignment of Menaquinone-specific isochorismate synthase showing a 4-amino-acid insertion identified in all members ofPasteurella sensu stricto. This insertion was not found in the homologues from any member of the genusPasteurella that was not part of the “sensu stricto” clade or any other member of theGammaproteobacteria. Sequence information for other CSIs specific toPasteurella sensu stricto is presented in Supplemental Figures  9–13 and their characteristics are summarized in Table 2
(
C
).


## 3.6. Molecular Signatures Specific for the GeneraAggregatibacter orMannheimia

The genusAggregatibacter was proposed as a novel taxonomic classification for a monophyletic cluster ofActinobacillusandHaemophilus species which branched distinctly from the “sensu stricto” members of their respective clades [21]. Similarly, the genusMannheimia was proposed as a novel classification for thePasteurella Haemolytica complex which did not branch withPasteurella sensu stricto in phylogenetic trees [7]. Currently other than branching in phylogenetic trees or relatedness in DNA-DNA hybridization studies, the members of the generaAggregatibacter orMannheimia do not share any single unique or defining biochemical or molecular characteristic that can differentiate them from all other bacteria [5, 82].In this study we have identified 4 CSIs that are unique molecular characteristics shared by all sequenced species of the genusAggregatibacter and another 4 CSIs which are uniquely found in all sequenced members of the genusMannheimia. Examples of CSIs specific to the sequenced members ofAggregatibacter andMannheimia are shown in Figure 5. A partial sequence alignment of anhaC family sodium:proton antiporter containing a 3-amino-acid insertion specific for all sequenced species of the genusAggregatibacter is shown in Figure 5(a) and a partial sequence alignment of a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specific for all sequenced species of the genusMannheimia is shown in Figure 5(b). In each case, the identified CSIs were only found in the sequenced members of the generaAggregatibacter orMannheimia and were absent from all other sequencedGammaproteobacteria. Partial sequence alignments additional CSIs specific for the generaAggregatibacter orMannheimia are provided in Supplemental Figures 14
–
19 and their characteristics are summarized in Tables 2
(
D
)
-
2
(
E
). These CSIs are the first discrete molecular characteristics which are unique for the generaAggregatibacter andMannheimia and support their observed monophyly in phylogenetic trees. Additionally, these CSIs could be useful targets for the development of PCR based diagnostic assays for the generaAggregatibacter andMannheimia which amplify the CSI containing DNA segment using the conserved flanking regions of the CSIs [83, 84].Figure 5
A partial sequence alignment of (a) anhaC family sodium:proton antiporter containing a 3-amino-acid insertion specific for all sequenced species of the genusAggregatibacter (b) a methyl-galactoside ABC transporter substrate-binding protein containing a 1-amino-acid deletion specific for all sequenced species of the genusMannheimia. In each case, the identified CSIs were only found in the sequenced members of the generaAggregatibacter orMannheimia and were absent from all other sequencedGammaproteobacteria. Sequence information for other CSIs specific toAggregatibacter orMannheimiais presented in Supplemental Figures  14–19 and their characteristics are summarized in Tables 2
(
D
) and 2
(
E
).
(a)
(b)


## 4. Conclusion

The generaActinobacillus, Haemophilus,andPasteurella, within the familyPasteurellaceae, are known to exhibit extensive polyphyletic branching. We have utilized molecular signatures and phylogenetic analyses to clarify the taxonomic boundary of these genera. We have been able to identify large clusters ofActinobacillus, Haemophilus,andPasteurella species which represent the “sensu stricto” members of these genera. We have identified 3, 7, and 6 unique molecular signatures which are specifically shared by the members of the generaActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto, respectively. The group specificity of the molecular signatures we have identified in this work is summarized in Figure 6 and their characteristics are briefly summarized in Table 2. Our comparative genomic analyses have not come across any CSIs that were unique characteristics of all sequenced members of the generaActinobacillus, Haemophilus,orPasteurella as currently defined, suggesting that the members of these genera that do not fall into the “sensu stricto” clusters should not be considered members of their respective genus.Figure 6
A summary diagram depicting the distribution of identified CSIs for genera within the familyPasteurellaceae.Examinations of phenotypic and biochemical characteristics do not provide a reliable means of assigning a novel isolate to the generaActinobacillus, Haemophilus,andPasteurella [18]. However, based upon the CSIs described in this work, it is now possible to demarcate the generaActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu stricto on the basis of the presence or absence of unique molecular signatures. It is important to note that the current analysis of CSIs is limited to the currently available genomic sequence data and may show slight variance as additional bacterial genomes are sequenced. However, earlier work on CSIs for other groups of bacteria provides evidence that the identified CSIs have strong predictive value and will likely be found in other members of these groups as more species are sequenced and novel species are isolated [74, 77, 85, 86]. The conserved nature of the sequence regions that contain these CSIs, in conjunction with their strong predictive value, makes CSIs promising targets for the development of highly specific diagnostic assays forActinobacillus sensu stricto, Haemophilus sensu stricto, Pasteurella sensu stricto,Aggregatibacter, andMannheimia [83, 84]. Additionally, further analysis of these genus specific CSIs should lead to the discovery of their functional role in their respective organisms and may provide important insights into novel distinguishing features of these groups of organisms.



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