Genome sequence of Haemophilus parasuis strain 29755

Haemophilus parasuis is a member of the family Pasteurellaceae and is the etiologic agent of Glässer’s disease in pigs, a systemic syndrome associated with only a subset of isolates. The genetic basis for virulence and systemic spread of particular H. parasuis isolates is currently unknown. Strain 29755 is an invasive isolate that has long been used in the study of Glässer’s disease. Accordingly, the genome sequence of strain 29755 is of considerable importance to investigators endeavoring to understand the molecular pathogenesis of H. parasuis. Here we describe the features of the 2,224,137 bp draft genome sequence of strain 29755 generated from 454-FLX pyrosequencing. These data comprise the first publicly available genome sequence for this bacterium.


Introduction
H. parasuis is an obligate pathogen of swine [1]. The bacterium is often carried in the nasal passages [2], but not the lungs [3], 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 [4][5][6][7][8] 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 [9], a class recognized as highly virulent and frequently isolated from respiratory and systemic sites [9,10]. Of the 15 recognized serovars, serovar 5 strains are isolated more frequently worldwide than any other [11]. 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 [12] (Table 1). A phylogenetic tree based on 16S ribosomal RNA sequences is depicted in Figure 1 for H. parasuis and related organisms. H. parasuis is a small, non-motile, rod-shaped bacterium [1] (Figure 2). The presence of a capsule is variable and may affect colony and cellular morphology [30]. Growth of the bacterium in vitro is dependent on the coenzyme nicotinamide adenine dinucleotide (NAD, or V factor) [31] but, in contrast to some other members of the genus, does not require porphyrins like hemin (X factor) [32]. 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 [1]. Temperature range mesophile (20°C-37°C) TAS [12] Optimum temperature 35°C-37°C TAS [12] Carbon source saccharolytic TAS [24] Energy source chemoorganotroph TAS [24] Terminal electron receptor Oxygen TAS [25] MIGS  [34] and the genome content is summarized in Table 3.

Growth conditions and DNA isolation
H. parasuis 29755 was grown from a frozen seed stock for two days under 5% CO2 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.  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

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 [35] to predict final location within the cell and assigned to COG functional categories (Table  4).

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). 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.