Introduction
============

*H. parasuis* is an obligate pathogen of swine \[[@r1]\]. The bacterium is often carried in the nasal passages \[[@r2]\], but not the lungs \[[@r3]\], of healthy pigs. Through unknown mechanisms some strains can spread systemically and may be isolated from the meninges, lungs, serosa, joints, and blood. *H. parasuis* strain 29755 (IA84-29755), though not the type strain, has been used extensively in a variety of investigations \[[@r4]-[@r8]\] and is the most fully characterized strain of the species. Originally cultured at Iowa State University from a pig exhibiting Glässer's disease, 29755 is a serovar 5 isolate \[[@r9]\], a class recognized as highly virulent and frequently isolated from respiratory and systemic sites \[[@r9],[@r10]\]. Of the 15 recognized serovars, serovar 5 strains are isolated more frequently worldwide than any other \[[@r11]\]. Strain 29755 has been used as a component of at least one commercially available *H. parasuis* vaccine (Suvaxyn M. hyo -- parasuis, Fort Dodge Animal Health).

Classification and features
===========================

The genus *Haemophilus* belongs to the *Gammaproteobacteria* and is classified in the family *Pasteurellaceae* \[[@r12]\] ([Table 1](#t1){ref-type="table"}). A phylogenetic tree based on 16S ribosomal RNA sequences is depicted in [Figure 1](#f1){ref-type="fig"} for *H. parasuis* and related organisms.

###### MIGS classification and general features of *H. parasuis* strain 29755.

  **MIGS ID**      **Property**                        **Term**                                **Evidence code**
  ------------- ----------------------------------- ---------------------------------------- ---------------------
                   Current classification              Domain *Bacteria*                       TAS \[[@r13]\]
                   Phylum *Proteobacteria*            TAS \[[@r14]\]                         
                   Class *Gammaproteobacteria*        TAS \[[@r15],[@r16]\]                  
                   Order *Pasteurellales*             TAS \[[@r15],[@r17]\]                  
                   Family *Pasteurellaceae*           TAS \[[@r18],[@r19]\]                  
                   Genus *Haemophilus*                TAS \[[@r20]-[@r22]\]                  
                   Species *Haemophilus parasuis*     TAS \[[@r20],[@r23]\]                  
                   Strain 29755                                                              
                   Serotype 5                                                                
                   Gram stain                          negative                                TAS \[[@r1]\]
                   Cell shape                          rods (pleomorphic)                      TAS \[[@r1]\]
                   Motility                            nonmotile                               TAS \[[@r1]\]
                   Sporulation                         non-sporulating                         TAS \[[@r1]\]
                   Temperature range                   mesophile (20°C-37°C)                   TAS \[[@r12]\]
                   Optimum temperature                 35°C-37°C                               TAS \[[@r12]\]
                   Carbon source                       saccharolytic                           TAS \[[@r24]\]
                   Energy source                       chemoorganotroph                        TAS \[[@r24]\]
                   Terminal electron receptor          Oxygen                                  TAS \[[@r25]\]
  MIGS-6           Habitat                             Host, swine upper respiratory tract     TAS \[[@r1]\]
  MIGS-6.3         Salinity                            1-1.5%                                  TAS \[[@r12]\]
  MIGS-22          Oxygen requirement                  facultative                             TAS \[[@r12]\]
  MIGS-15          Biotic relationship                 obligate pathogen of swine              TAS \[[@r1]\]
  MIGS-14          Pathogenicity                       mild to severe                          TAS \[[@r1]\]
  MIGS-4           Geographic location                 Iowa                                    NAS
  MIGS-5           Sample collection time              1970s                                   NAS
  MIGS-4.1         Latitude                            not reported                          
  MIGS-4.2         Longitude                           not reported                          
  MIGS-4.3         Depth                               not reported                          
  MIGS-4.4         Altitude                            not reported                          

Evidence codes - NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project \[[@r26]\]

![Phylogenetic tree based on 16S rRNA of *H. parasuis* 29755 and type strains of some closely related species and other genera within the *Pasteurellaceae*. Also included is the only additional *H. parasuis* strain for which a genome sequence has been reported, SH0165. The tree was generated with the tree-builder available from the Ribosomal Database Project\[[@r27]\] using the Weighbor (weighted neighbor-joining) algorithm \[[@r28]\] with Jukes-Cantor distance correction \[[@r29]\]. Numbers to the left of branches indicate the percentage of trees in which each branch was represented in 100 replicates. An *E. coli* type strain was used as an outgroup.](sigs.2245029-f1){#f1}

*H. parasuis* is a small, non-motile, rod-shaped bacterium \[[@r1]\] ([Figure 2](#f2){ref-type="fig"}). The presence of a capsule is variable and may affect colony and cellular morphology \[[@r30]\]. Growth of the bacterium *in vitro* is dependent on the coenzyme nicotinamide adenine dinucleotide (NAD, or V factor) \[[@r31]\] but, in contrast to some other members of the genus, does not require porphyrins like hemin (X factor) \[[@r32]\]. Plating on Casman Agar Base (BBL) supplemented with 1% (w/v) NAD (Sigma) and 5% GIBCO filtered horse serum (Invitrogen) or on chocolate agar produces small, translucent colonies that appear within 24 hours and reach full size in approximately two days. Colonies are nonhemolytic when grown on blood agar \[[@r1]\]. *H. parasuis* grows under normal atmosphere at 37°C, although added humidity and 5% CO~2~ may improve growth.

![Scanning electron micrograph of *H. parasuis* 29755](sigs.2245029-f2){#f2}

Genome sequencing and annotation
================================

Genome project history
----------------------

*H. parasuis* strain 29755 was selected for sequencing because it has long been used in the study of Glässer's disease. Pyrosequencing (454 Life Sciences) was performed at the State University of New York, University at Buffalo Center of Excellence in Bioinformatics and Life Sciences. The draft genome sequence is deposited in GenBank (NZ_ABKM00000000). Summary project information is shown in [Table 2](#t2){ref-type="table"} according to the Minimum Information about a Genomic Sequence (MIGS) recommendations \[[@r34]\] and the genome content is summarized in [Table 3](#t3){ref-type="table"}.

###### Genome sequencing project information

  **MIGS ID**      **Property**                 **Term**
  ------------- ---------------------------- ------------------------------------------
  MIGS-28          Libraries used               one 454 pyrosequence standard library
  MIGS-29          Sequencing platforms         454 (FLX)
  MIGS-30          Assemblers                   Newbler
  MIGS-31          Finishing quality            draft
  MIGS-31.2        Fold coverage                28×
  MIGS-32          Gene calling method          Glimmer, GeneMark \[[@r33]\]
                   Genome Database release      February 14, 2008
                   Genbank ID                   NZ_ABKM00000000
                   Genbank Date of Release      February 14, 2008
                   GOLD ID                      -
                   Project relevance            food animal pathogenesis

###### Genome statistics

  **Attribute**                  **Value**      **% of total^a^**
  --------------------------- -------------- ----------------------
  Size (bp)                      2,224,137      100.0%
  G+C content (bp)               867,413        39.0%
  Coding region (bp)             1,890,516      85.0%
  Total genes                    2,309          100.0%
  RNA genes                      58             2.5%
  Protein-coding genes           2,244          97.2%
  Pseudogenes                    none^b^        0.0%
  Genes in paralog clusters      nd^c^          -
  Genes assigned to COGs         1,926          83.4%
  PSORT cytoplasmic              1,181          50.4%
  PSORT extracellular            5              0.2%
  PSORT outer membrane           51             2.2%
  PSORT periplasmic              52             2.2%
  PSORT unknown                  1,055          45.0%

^a^ Based either on the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

^b^ Based on preliminary analysis the of draft genome

^c^ nd = not determined

Growth conditions and DNA isolation
-----------------------------------

*H. parasuis* 29755 was grown from a frozen seed stock for two days under 5% CO~2~ at 37°C on Casman Agar Base (BBL) supplemented with 1% (w/v) NAD (Sigma) and 5% GIBCO filtered horse serum (Invitrogen). Following growth, a single colony was used to inoculate 5 ml of brain-heart infusion medium supplemented with 10 μg/ml NAD and 10 μg/ml hemin (sBHI) and the culture was incubated overnight at 37°C and 185 rpm. The next day, 2 ml of the culture were added to 100 ml of sBHI and the bacterium was again allowed to grow overnight to stationary phase at 37°C and 185 rpm. Bacterial cells were pelleted by centrifugation at 4000 × *g* for 10 minutes. The pellet was resuspended and used as the source of genomic DNA purified with the QIAGEN Blood & Cell Culture DNA Kit, as recommended by the manufacturer. The final preparation contained 1.12 μg/ul genomic DNA as determined by UV absorption spectrometry.

Genome sequencing and assembly
------------------------------

Library preparation yielded 9.65 × 10^8^ molecules/μl of DNA with a mean size of approximately 600 nucleotides, as determined with a RNA6000 Pico chip on an Agilent 2100 Bioanalyzer. Emulsion PCR was performed at a concentration of 2 molecules per bead. Following sequencing, contigs were assembled using the 454 Newbler assembler.

Genome annotation
-----------------

Genes were identified manually using GeneMark and automatically using Glimmer as part of the NCBI draft genome submission pipeline. Translated protein sequences were analyzed using PSORTb v.2.0 \[[@r35]\] to predict final location within the cell and assigned to COG functional categories ([Table 4](#t4){ref-type="table"}).

###### Number of genes associated with the general COG functional categories

  **Code**     **Value**     **%age**^a^      **Description**
  ---------- ------------- --------------- -----------------------------------------------------------------
  J            168           6.55             Translation
  A            1             0.03             RNA processing and modification
  K            127           4.96             Transcription
  L            166           6.48             Replication, recombination and repair
  B            0             0.00             Chromatin structure and dynamics
  D            33            1.29             Cell cycle control, mitosis and meiosis
  Y            0             0.00             Nuclear structure
  V            32            1.25             Defense mechanisms
  T            48            1.87             Signal transduction mechanisms
  M            134           5.23             Cell wall/membrane biogenesis
  N            16            0.62             Cell motility
  Z            0             0.00             Cytoskeleton
  W            24            0.94             Extracellular structures
  U            75            2.93             Intracellular trafficking and secretion
  O            101           3.94             Posttranslational modification, protein turnover, chaperones
  C            115           4.49             Energy production and conversion
  G            139           5.42             Carbohydrate transport and metabolism
  E            175           6.83             Amino acid transport and metabolism
  F            57            2.22             Nucleotide transport and metabolism
  H            97            3.78             Coenzyme transport and metabolism
  I            43            1.68             Lipid transport and metabolism
  P            116           4.53             Inorganic ion transport and metabolism
  Q            25            0.96             Secondary metabolites biosynthesis, transport and catabolism
  R            234           9.13             General function prediction only
  S            197           7.69             Function unknown
  \-           440           17.16            Not in COGs

^a^ Based on the total number of protein coding genes in the annotated genome

Genome properties
=================

The draft genome is 2,224,137 bp and is likely comprised of one circular chromosome with a G+C content of approximately 39% ([Figure 3](#f3){ref-type="fig"}). For display, contigs were assembled end-to-end with twenty "N" bases between contigs. Orientation and order of contigs will change when the genome sequence is closed.

![Graphical circular map of the *H. parasuis* 29755 draft pseudogenome. From the outside to the center: open reading frames (ORFs) on the forward strand (one ring for each reading frame), start and stop codons for forward and reverse strands, ORFs on the reverse strand, GC content, and GC skew. The map was generated using CGView Server \[[@r36],[@r37]\].](sigs.2245029-f3){#f3}

The authors wish to thank David Alt, USDA/ARS/National Animal Disease Center for technical advice and the State University of New York, University at Buffalo Center of Excellence in Bioinformatics and Life Sciences for performing pyrosequencing. This work was supported, in part, by grants from the NIH/NCRR (D.W. Dyer, Grant \#P2PRR016478), National Pork Board (G.J. Phillips and D.W. Dyer) and Iowa Healthy Livestock Initiative (G.J. Phillips and K.B. Register).
