Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.
Citation: Xu Z, Yue M, Zhou R, Jin Q, Fan Y, Bei W, et al. (2011) Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China. PLoS ONE 6(5): e19631. https://doi.org/10.1371/journal.pone.0019631
Editor: Baochuan Lin, Naval Research Laboratory, United States of America
Received: November 23, 2010; Accepted: April 11, 2011; Published: May 17, 2011
Copyright: © 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (30901075, 30970109), National Programs for High Technology Research (No. 2010AA10A202) and Program for Changjiang Scholars and Innovative Research Team in University (IRT0726). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The Gram-negative bacterium Haemophilus parasuis is a strictly swine pathogen. It is a non-motile, pleomorphic NAD-dependent coccobacillus belonging to the family Pasteurellaceae of the γ-proteobacteria . As a commensal colonizing the upper respiratory tract of pigs, H. parasuis is also an opportunistic etiologic agent that can cause serious systemic disease, namely Glässer's disease, which is characterized by fibrinous polyserositis, arthritis and meningitis, or acute pneumoniae and acute septicaemia . In recent years, infections caused by this bacterial pathogen have led to great economic losses in the pig industry worldwide.
Based on the heat-stable antigens and the gel diffusion test, 15 serovars of H. parasuis have been classified, with apparent differences in virulence . However, there are a large number of non-typeable H. parasuis strains exhibiting high heterogeneity at the molecular level , perhaps due to recombination or lateral gene transfer. H. parasuis serovar 4 predominates in North American herds, whereas serovar 5 and non-typeable strains are commonly isolated in many European countries , . In China, the most prevalent serovars are 4, 5 and 13 .
To date, many potential virulence-associated factors investigated in the family Pasteurellaceae have been identified in H. parasuis, including lipopolysaccharide (LPS), capsular polysaccharide, fimbriae, outer membrane proteins, neuraminidase and iron acquisition systems , –. However, the molecular basis underlying these candidate virulence factors is yet to be fully elucidated.
Currently, the complete genome sequences of eight strains from four species within the genus Haemophilus are publicly available (up to June, 2010). These include H. parasuis SH0165 (GenBank accession no. CP001321), H. ducreyi 35000HP (AE017143), H. influenzae Rd KW20 (L42023), 86-028NP (CP000057), PittEE (CP000671), PittGG (CP000672), Histophilus somni 129PT (CP000436) and 2336 (CP000947). Among these, H. influenzae Rd and H. somni 129PT are both nonpathogenic strains of human and bovine origin, respectively, and H. ducreyi 35000HP causes chancroid, a human sexually transmitted disease , . In this study, we carried out a comprehensive genomic characterization of H. parasuis SH0165, a high virulent serovar 5 strain recovered from the lung of a diseased piglet in North China. Comparisons of the genomic components between H. parasuis SH0165 and other representative species within the family Pasteurellaceae were performed in detail. Our work focused on identifying the particular genes/pathways that play a role in carbon metabolism, natural competence, virulence and host colonization of H. parasuis.
Bacterial strain and genome sequencing
H. parasuis strain SH0165 was isolated in 2001 from the lung of a diseased piglet in North China and was identified as serovar 5. This bacterial strain was cultured on tryptic soy agar (TSA) plates supplemented with 5% bovine serum and 10 µg/ml nicotinamide adenine dinucleotide (NAD). Bacterial DNA was extracted and sequenced using a shotgun sequencing strategy, as described previously .
Glimmer3 , tRNASCAN-SE  and RNAmmer  were used to predict protein coding sequences (CDSs), tRNAs and rRNAs, respectively. BLASTP  was used for automated annotation based on sequence similarity in conjunction with searching the CDS set against the cluster of orthologous groups (COG) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Pfam protein families database and NCBI non-redundant protein database. A genome comparative circular map was implemented using the program CGview . The sequenced draft genome of H. parasuis serovar 5 strain 29755 (NZ_ABKM00000000) was aligned to the complete genome of H. parasuis strain SH0165 (CP001321) using BLASTN (minimum sequence identity of 95% and expected threshold of 1e−5). Short DNA repeats in the genome sequences were searched with a Perl script repeat_finder (http://folk.uio.no/stephanf/repeat_finder.html) and graphical representations of the patterns in sequence conservation were created using WebLogo . Protein transmembrane helices were predicted by the package TMHMM 2.0 .
Ortholog identification and phylogenetic reconstruction
All protein sequences from the complete genomes of H. somni 129PT (CP000436), H. influenzae Rd (L42023), H. ducreyi 35000HP (AE017143), Actinobacillus pleuropneumoniae JL03 (CP000687), A. succinogenes 130Z (CP000746), Aggregatibacter aphrophilus NJ8700 (CP001607), A. actinomycetemcomitans D11S-1 (CP001733), Pasteurella multocida Pm70 (AE004439) and Mannheimia succiniciproducens MBEL55E (AE016827), were retrieved from the GenBank database. Protein sequences from the draft genome assemblies of H. parasuis strain 29755 were also retrieved. In this study, orthologous pairs of proteins between the CDS sets of two genomes were defined if protein identity was above 45%, alignment coverage above 70% and E_value below 1e−20. Orthologs present in the complete genomes of ten Pasteurellaceae species were retrieved. These protein sequences were aligned using the program MUSCLE 3.6  and 727 alignments were then concatenated into a large alignment of 245,554 amino acids. A neighbor joining tree was reconstructed in the software MEGA 4  via the bootstrap test of 1,000 replicates.
Results and Discussion
Genome structure and general features
H. parasuis SH0165 contains a single, circular chromosome that is 2,269,156 base pairs (bp) in length (Figure 1). The chromosome encodes 2,031 protein-coding genes (192 pseudogenes not included herein), 56 tRNA genes and 20 rRNA genes on six ribosomal rRNA operons. Global characterizations of the H. parasuis SH0165 genome were summarized and compared to those of strains 129Pt of H. somni, Rd of H. influenzae, 35000HP of H. ducreyi, JL03 of A. pleuropneumoniae and Pm70 of P. multocida (Table 1). In comparison with the other Haemophilus spp., H. parasuis has a considerably larger chromosome, which is almost equal in size to those of P. multocida and A. pleuropneumoniae. The overall G+C content of the H. parasuis SH0165 genome was 40.0%, a bit higher than those of the other Haemophilus genomes. Pairwise nucleotide sequence alignments between the complete genome of H. parasuis SH0165 and the contigs of H. parasuis serovar 5 strain 29755 indicated that approximately 2,003 kb of sequence (above 95% identity) was shared by both strains, accounting for 88% of the total length of the SH0165 genome. There were 1,714 orthologous pairs of protein-coding genes between both H. parasuis strains of serovar 5, and H. parasuis strain SH0165 has 317 genes absent in strain 29755. It is worth noting that one third (104 genes) of these unique genes were phage-related. The H. parasuis SH0165 genome had about 198 CDSs encoding proteins involved in phage functions or phage islands, accounting for approximately 10% of the total CDSs. The majority of phage-related genes were mainly located in three phage islands designated HP-P1 (68.4 kb ranging from HAPS0536 to HAPS0638), HP-P2 (38.8 kb, HAPS0859–0910) and HP-P3 (9 kb, HAPS1147–1160). It has been suggested that the integration of phage elements, as a strategy of horizontal gene transfer, play a potentially important role in genetic diversity and virulence variations in many bacteria . The phage-related genes found in the H. parasuis SH0165 genome may be also a putative contributor to virulence and inheritance differences.
Circles range from 1 (the outermost circle) to 8 (the innermost circle). The outer two circles show protein-coding genes on the forward and reverse strands in SH1605, colored according to COG categories. All genes are colored based on biological functions: maroon for translation, ribosomal structure and biogenesis; tomato for RNA processing and modification; navy for transcription; purple for DNA replication, recombination and repair; light brown for cell division and chromosome partitioning; gold for defense mechanisms; light purple for signal transduction; teal for cell envelope biogenesis and outer membrane; chocolate for intracellular trafficking, secretion and vesicular transport; aqua for posttranslational modification, protein turnover and chaperones; olive for energy production and conversion; lime for carbohydrate transport and metabolism; green for amino acid transport and metabolism; fuchsia for nucleotide transport and metabolism; light pink for coenzyme metabolism; red for lipid metabolism; orange for inorganic ion transport and metabolism; yellow for secondary metabolites biosynthesis, transport and catabolism; gray for general functional prediction; silver for function-unassigned conserved proteins; and black for unknown proteins not in the COG collection. The third circle shows the coordinates of BLAST hits of the H. parasuis SH1605 complete genome against genomic contigs of H. parasuis strain 29755 and these are colored in maroon. Fourth circle, insertion sequence elements in green. Fifth circle, tRNA genes in red. Sixth circle, rRNA operons in blue. Seventh circle, G+C content. Eighth circle, GC skew plot [(G−C)/(G+C); green indicates values >0; purple indicates values <0].
Genome-wide comparisons of orthologous gene pairs between H. parasuis and other organisms within the family Pasteurellaceae showed that H. parasuis is more closely related to A. pleuropneumoniae (1,341 pairs of orthologs), which also colonizes the upper respiratory tract of pigs , but is more distantly related to the other three Haemophilus species, with only 1,095 orthologs found in H. influenzae (Table 2). Phylogenetic tree reconstruction based upon 727 conserved coding genes demonstrated that H. parasuis, A. pleuropneumoniae, and H. ducreyi were grouped into a subclade (Figure 2). However, the evolutionary relationship between H. parasuis and the strict human pathogen H. ducreyi is less obvious , as only 1,096 orthologs were identified in the genomes of both species. This may be due to adaptation of the two pathogens to different host species and tissues. These findings may indicate that, phylogenetically, the swine pathogens H. parasuis and A. pleuropneumoniae probably derive from a recent common ancestor and their adaptations reflect uniform host niches.
Tree derived from 727 concatenated, conserved protein sequences in the complete genomes of 10 Pasteurellaceae species. The scale indicates the number of substitutions per residue. Node support after 1,000 bootstrap replicates is shown.
Carbon source utilization
H. parasuis is a facultative anaerobe which possesses metabolic pathways of both fermentation and respiration for energy generation . Two kinds of sugar transport systems were identified in the H. parasuis SH0165 genome. We identified a predicted set of genes encoding ATP-binding cassette (ABC) transport complexes involved in the utilization of distinct sugars, including ribose (rbsDACB, HAPS1629–1632; rbsACB, HAPS1727–1725), galactose (mglBAC, HAPS0442–0444) and maltose (malEFGK, HAPS0236–0234, HAPS0237) (Figure 3). H. parasuis SH0165 also possesses genes encoding phosphotransferase systems (PTS), which can be devoted to the uptake of other sugars, including glucose (ptsHI-crr, HAPS0960–0958; ptsG, HAPS2092), fructose (ptsEII, HAPS0193–0189), sucrose (ptsB, HAPS1008) and mannose (manXYZ, HAPS1732–1730). These confirmed genotypes support the previously observed biochemical patterns of sugar fermentation in H. parasuis .
Genes encoding key enzymes and functional proteins involved in the metabolic pathways are illustrated while the corresponding coding sequences (CDSs) indicated by HAPS numbers are list in Table S1 in the supplemental material.
Complete sets of genes coding for enzymes involved in glycolysis and gluconeogenesis, the tricarboxylic acid (TCA) cycle, as well as the pentose phosphate pathway, were identified in the SH0165 genome (Figure 3). The Entner–Doudoroff pathway, an alternative bypass of classical glycolysis, was not encoded in the H. parasuis SH0165 genome, as genes edd and eda encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phosphate aldolase, respectively, are absent. By contrast, H. somni 129PT and H. influenzae Rd both have an active Entner–Doudoroff pathway responsible for the conversion of gluconate-6-phosphate to pyruvate . In addition, H. parasuis SH0165 possessed an enzyme (307 amino acids, aa), encoded by the rbsK2 (HAPS1688) gene, that was homologous to the fructokinase CscK (307 aa) of Escherichia coli EC3132, which can catalyze the phosphorylation of fructose to fructose-6-phosphate . Rbsk2 is capable of metabolizing fructose as an alternative start point to glycolysis. However, H. influenzae Rd, H. somni 129PT and H. ducreyi 35000HP all lack this gene. Five H. parasuis genes coding for enzymes involved in the conversion of pyruvate were identified, which are necessary for ATP production through lactic acid fermentation (ldhA, HAPS2117; dld, HAPS1875), and for the generation of formate (pflB, HAPS0155) and acetate (pta, HAPS0393; ackA, HAPS0392). Besides fermentation, H. parasuis may conduct the entire oxidation of glucose derivatives aerobically to release energy through an intact TCA system (Figure 3). In contrast, several other sequenced species in the genus Haemophilus, e.g., H. influenzae, H. ducreyi and H. somni, have been found to harbor deficiencies in the TCA cycle, as they are missing one or three genes coding for citrate synthase, aconitase and isocitrate dehydrogenase . In addition, genes aceA and aceB encoding isocitrate lyase and malate synthase, respectively, which code for key enzymes involved in the glyoxylate shunt, are absent in H. parasuis SH0165. Moreover, all known sequenced genomes of Haemophilus spp. are lacking the entire glyoxylate bypass.
H. parasuis may perform not only aerobic respiration but also anaerobic respiration, as approximately 61 genes involved in the branched electron respiration transport chains were identified in the SH0165 genome (Table S1). Besides cytochrome D ubiquinol oxidase encoded by cydAB (HAPS0067, 0068), which is used for aerobic respiration in the presence of oxygen, H. parasuis SH0165 possesses an intact napFDAGHBC (HAPS1796–1790) operon encoding a putative periplasmic nitrate reductase responsible for the reduction of terminal electron acceptor nitrates in anaerobic environments , .
To effectively control gene expression in response to environmental stimuli, H. parasuis SH0165 encodes about 94 genes related to regulatory functions (Table S2). Genes encoding conserved global regulatory proteins, such as Crp (HAPS2043), CyaA (HAPS0993), Fnr (HAPS0167), ScrR (HAPS1203), Mlc (HAPS0390) and CsrA (HAPS0464), are present and intact in the SH0165 genome. These regulators are able to play a role in activating or repressing the transcription of genes involved in carbon metabolism or other central metabolisms , . In addition, three predicted two-component signal transduction systems, encoded by cpxAR, arcAB, and qseBC, were also identified in the genome of H. parasuis SH0165. Two-component systems are known to mediate adaptive responses to various environmental signals . For instance, under anaerobic conditions, the ArcA-ArcB system may upregulate genes for anaerobic respiration while downregulate genes for aerobic respiration and fermentation , .
H. parasuis requires supplementation of the culture media with NAD (V factor) for growth, but NAD is not necessary for the in vitro growth of H. somni or H. ducreyi . Corresponding to these phenotypic differences, gene nadV, which encodes the nicotinamide phosphoribosyltransferase responsible for NAD salvage, is present in the genomes of H. somni and H. ducreyi (HS0002, HD1447 and HD1455) , , but is absent from the SH0165 genome. This is the first time that the NAD-dependent growth of H. parasuis can be explained genetically .
Unlike H. influenzae and H. ducreyi, H. parasuis can grow well in vitro without requiring additional supplementation of iron porphyrin (X factor) . This is because the heme biosynthetic pathway is present and intact in H. parasuis SH0165. An entire set of genes encoding enzymes that are responsible for the conversion of L-glutamate to protoheme were identified in our study (Table S3). Notably, H. influenzae Rd and H. ducreyi 35000HP were missing the main enzymes necessary for heme biosynthesis, but the animal-originated members within the Pasteurellaceae family, e.g. H. parasuis, H. somni, A. pleuropneumoniae and P. multocida, all had a complete biosynthetic pathway for the production of the porphyrin ring. Two hemN genes (HAPS0238 and 0482) encoding coproporphyrinogen III oxidases (EC: 126.96.36.199) were identified in H. parasuis SH0165. Despite the identity between HemN1 and HemN2 only being 23%, they both belong to the HemN_C (PF06969) family whose members catalyze the conversion of coproporphyrinogen-III to protoporphyrinogen-IX .
Otherwise, the SH0165 genome encodes almost all indispensable enzymes involved in the biosynthesis of amino acids, nucleotides, fatty acids and cofactors. The catalase encoded by hktE (HAPS2238) may support a catalase-positive phenotype . It is worth noting that genes ureABC encoding urease are all absent from the H. parasuis SH0165 genome, explaining the urease-negative characteristic of this species .
Many bacteria are competent, indicating that they have the ability to actively acquire extracelluar DNA from the environment . In comparison with the competence genes in H. influenzae and Mannheimia haemolytica , , the H. parasuis SH0165 genome also contains homologous genes encoding proteins involved in the assembly of the DNA uptake machinery. Outer membrane proteins ComE (HAPS2289) and PilA (HAPS2013) play a potential role in binding liberated DNA segments at the cell surface . Orthologs of proteins that function in the transport of DNA across the cell membrane, periplasmic space, then into the cytoplasm, include lipoprotein ComL (HAPS0926), periplasmic protein ComF (HAPS1497) and cytoplasmic membrane proftein ComEA (HAPS0844) . Additionally, several cytoplasmic competence proteins coding genes were identified in the H. parasuis SH0165 genome, including comA (HAPS2285), dprA (HAPS1573) and comM (HAPS1740) . These genetic components may contribute to the natural competence of H. parasuis .
It has been shown that Pasteurellaceae species preferentially take up short DNA sequences, called uptake signal sequences (USS), that are overrepresented in their respective genomes . In H. influenzae, the highly redundant USS has the sequence AAGTGCGGT, and this is also the most common uptake sequence in the genomes of H. somni, A. actinomycetemcomitans, P. multocida and M. succiniciproducens . However, in the genome sequence of H. parasuis SH0165, the most frequent nine-mer repeat is ACAAGCGGT, being present in 523 copies, whereas only 109 copies of the H. influenzae core USS were identified in H. parasuis. Moreover, the core USS of H. parasuis is identical to the most common nine-mer repeats previously identified in A. pleuropneumoniae and M. haemolytica . Figure 4 shows the 50-bp consensus sequences flanking the core USSs in the H. parasuis SH0165 genome. Two AT-rich motifs were observed downstream of the H. parasuis USSs and similar patterns of sequence conservation have been found in M. haemolytica and A. pleuropneumoniae, further confirming that these three microorganisms belong to the same evolutionary subclade , .
Adherence and secretion
Fimbriae (namely pili) are a common virulence factor that mediate bacterial adherence to mucosal epithelia . Fimbria-like structures have been observed on the surface of H. parasuis after in vivo passage . As previously reported, a gene cluster pilABCD coding for type IV fimbriae has been identified in a number of Gram-negative pathogens in the genera of Haemophilus, Actinobacillus, Pseudomonas, Vibrio and others , . H. parasuis SH0165 also possesses four type IV fimbrial genes encoding the major structural unit PilA (HAPS2013) and three biogenesis proteins PilBCD (HAPS2011–2009) for mediating bacterial adherence. The proteins encoded by these CDSs share 68%, 68%, 50% and 44% identity with the ApfABCD proteins of A. pleuropneumoniae HK361, respectively . In addition to PilABCD, H. parasuis SH0165 also encodes a protein (HAPS2143, 22.3 kDa) that shares 54% similarity with Pseudomonas aeruginosa PilF (22.4 kDa), which is involved in type IV fimbrial biogenesis and twitching motility .
Genes coding for proteins that constitute the classical Sec and Tat secretion systems were identified in the genome of H. parasuis SH0165 (Table 3), which are required for protein secretion and trafficking. Ten paralog genes vtaA coding for virulence-associated trimeric autotransporters of the type V secretion system were also found in SH0165, the products of which are generally exported through the cytoplasmic membrane via the Sec system . Recent studies have indicated that the H. parasuis vtaA genes reveal diversity and the expressed products are good candidate immunogens , . Furthermore, three potential autotransporters related to the type V protein secretion pathway were encoded in the SH0165 genome, including two EspP proteins (HAPS1378, 771 aa; HAPS1381, 780 aa) and AidA (HAPS0753, 858 aa), all of which have a C-terminal translocator domain (PF03797) responsible for the transport of the N-terminal passenger domain across the outer membrane . The amino acid sequence identity between both EspP proteins was high (73%), indicating that they may be encoded by duplicate copies of the same gene.
Biosynthesis of surface polysaccharide
LPSs are the primary structural and functional components of the Gram-negative bacterial outer membrane, which can be recognized and targeted by the mammalian immune system . Typical LPS molecules consist of three covalently linked biochemical moieties: the lipid A, the core oligosaccharide and the major surface-exposed O-specific antigen (known as the O polysaccharide). The O polysaccharide is composed of many repeats of an oligosaccharide unit (O unit) and plays a vital role in bacterial adherence, invasion and immune evasion . The LPSs of H. parasuis, also termed lipo-oligosaccharides (LOSs) in this bacterium, are considered a virulence factor associated with disseminated intravascular coagulation and thrombosis , .
All genes coding for enzymes involved in the biosynthesis of LOS were identified in the SH0165 genome (Table S4). Unsurprisingly, according to sequence comparisons and function assignment, genes essential for the synthesis of lipid A (lpxC, kdsA, lpxB, kdsB, lpxH, lpxK, lpxD, lpxA, kdtA, lpxM, kdsC and lpxL) and core oligosaccharide (rfaE, rfaF, rfaD, lgtF and gmhA) are present and highly conserved among the genus Haemophilus. Furthermore, a 13,964 bp genomic region that is likely associated with O-antigen biogenesis was predicted in the H. parasuis SH0165 genome. Twelve putative CDSs were identified within this potential O-antigen region located between HAPS0039 and HAPS0052, and all of these share the same transcriptional orientation (Figure 5). Like many other Gram-negative bacteria, the H. parasuis O-antigen gene cluster had a G+C content (32.8%) lower than that of the SH0165 genome (40.0%). Nine of these CDSs could be divided into three categories according to their functional roles in the biogenesis of O-antigen : sugar synthesis-related genes (neuA1 and wbgX); glycosyltransferase genes (lsgB, wcwK, wcfQ and wbgY); and O unit processing genes wzx, wzy and wzz involved in the assembly of O-polysaccharide.
The coding sequences (CDSs) are drawn to scale, with 1 kb increments indicated.
In general, O-unit-processing enzymes (Wzx, Wzy and Wzz) in diverse Gram-negative bacteria show considerable sequence variation but share conserved topologies in membrane spanning regions . The wzx (HAPS0041) gene of H. parasuis encodes an O-antigen flippase (398 aa) which has 12 predicted transmembrane helices and shares 48% identity and 69% similarity with a Wzx homologue (404 aa) from the Shigella boydii O-antigen gene cluster . Although BLASTP searches did not detect any significant homologues of the product of HAPS0043, it was predicted to be an inner-membrane protein with 10 transmembrane helices that contains a large periplasmic loop of 44 amino acid residues (Figure 6), a topology typical of all known O-antigen polymerases , . Thus, we propose that HAPS0043 of H. parasuis is the O-antigen polymerase gene, wzy. More experimental works are still required to verify the function of H. parasuis Wzy in the O-antigen-processing process.
A. HAPS0041 (APJL1491, wzx), encoding an O-antigen flippase; B. HAPS0043 (APJL1490, wzy), encoding a putative O-antigen polymerase; C. HAPS0051 (APJL1485, wzz), encoding an O-antigen chain length determining protein. All the amino acid sequences were retrieved from the genomes of H. parasuis SH0165 and A. pleuropneumoniae JL03. The posterior probability of the transmembrane helix, intracellular side and extracellular side are indicated in green, red and blue, respectively.
As is well known, sialylated oligosaccharide units in bacterial polysaccharides play a role in evading the host immune response by mimicking the glycolipid components of mammalian tissues . A CMP-NeuNAc synthetase (N-acetylneuraminic acid cytidylsynthetase) (226 aa) encoded by the neuA1 (HAPS0040) gene of H. parasuis SH0165 shares 63% identity and 81% similarity with NeuA (229 aa) of H. ducreyi 35000 . Furthermore, LsgB (322 aa), encoded by HAPS0042 of H. parasuis, exhibits about 51% similarity and 31% identity with Lst (371 aa) of Neisseria meningitidis and has a glycosyltransferase family 52 domain (PF07922) bearing alpha-2,3-sialytransferase activity. Lst encodes a CMP-NeuNAc transferase that is responsible for the linkage α-NeuNAc-(2–3)-Gal . NeuA and Lst have been reported to be essential for the sialic acid-containing glycoform of H. ducreyi LOS . Therefore, the identification of these two homologues in H. parasuis may indicate that this organism can synthesize a sialylated LPS glycoform. In addition, a putative nucleotide sugar aminotransferase WbgX encoded by HAPS0046 exhibits 85% similarity and 72% identity to the Shigella sonnei homologue that is involved in the synthesis of 2-acetamino-4-amino-2,4,6-trideoxy-D-galactose (4n-FucNAc) .
The H. parasuis SH0165 O-antigen gene cluster encodes four glycosyltransferases that sequentially add diverse sugar residues to make an expected four-sugar repeating O-unit. Besides LsgB, WcfQ (272 aa) encoded by HAPS0045 belongs to the glycosyl transferase family 2 (PF00535, E_value = 2×10−18) whose members can transfer different sugar donors to receptors. H. parasuis WcfQ is also 58% similar to the putative glycosyltransferase WcfQ (268 aa) involved in the type A capsular polysaccharide biosynthesis of Bacteroides fragilis 9343 . Gene wcwK (HAPS0044) encodes an enzyme (343 aa) that shares 35% identity (53% similarity) with a capsular polysaccharide phosphotransferase (335 aa) found in Streptococcus pneumoniae . WbgY encoded by HAPS0047 has 79% similarity and 59% identity to a bacterial sugar transferase involved in the first step of Plesiomonas shigelloides O17 O-antigen synthesis .
The deduced protein products (CapD, Wza, Ptp and Wzz) of the last four genes (HAPS0048–0051) in the H. parasuis SH0165 O-antigen chain cluster are homologous to the proteins WbfY, Wza, Wzb and Wzc encoded in the Vibrio vulnificus group 1 capsular polysaccharide operon, sharing 52%, 53%, 57% and 50% aa identity, respectively . It has recently been shown that CapD and WbgY are also virulence-associated genes of H. parasuis through the suppression subtractive hybridization of a highly virulent and a nonvirulent strain .
Another gene cluster involved in LOS biosynthesis was identified in the H. parasuis SH0165 genome, which also has a low G+C content (28.4%), indicating that this DNA region may originate from a species other than H. parasuis. This cluster contains six putative sugar transferase genes, lbgB, rfaF2, lbgA, rfaG, wabH and lpsA, which appear to be unique to H. parasuis, with most of them being absent from other species within the genus Haemophilus (Table S4). The products encoded by these CDSs belong to the glycosyltransferase family 1 (HAPS1021, 1022), 9 (HAPS1017, 1019) and 25 (HAPS1020, 1023), respectively.
Interestingly, nine genes were identified in the H. parasuis SH0165 genome that encode enzymes homologous to those encoded by the wec genes associated with the synthesis of Enterobacterial Common Antigen (ECA), a complex cell surface glycolipid found in all Gram-negative enteric bacteria . Unlike the wec gene cluster in the E. coli K12 chromosome , the H. parasuis ECA-like related genes, wecA, wecB, wecC, rmlB, wecD, wecE, wecF, wzyE and wecG, are dispersed throughout the SH0165 chromosome (Table S4). The identification of these genes provides genetic evidence that H. parasuis may produce a putative ECA-like glycoconjugate. It is worth mentioning that the wec homologues are also present and linked in a cluster in H. ducreyi 35000HP .
A number of enzymes, including neuraminidase and proteases, may be important for the virulence of H. parasuis. The gene nanH (HAPS1616) coding for a potential virulence factor, neuraminidase (sialidase), was identified in the SH0165 genome and its product may function in H. parasuis colonization . The H. parasuis SH0165 genome contains approximately 37 genes encoding different proteases that may contribute to virulence (Table S5). The majority of protease-coding genes are conserved among members of the Pasteurellaceae, perhaps due to their role in proteolysis for protein quality control, but several other proteases (encoded by HAPS0648, 1928 and 2032) may be unique to H. parasuis. A clpPX (HAPS2006, 2007) operon encodes the structural subunits of the ATP-dependent Clp protease which proteolysis-function has been reported to be pivotal to the secretion processes of Gram-negative pathogens . The gene lon (HAPS1011) encodes a cytoplasmic serine protease (800 aa) that shares 70% identity with Salmonella enterica serovar Typhimurium Lon protease (784 aa), a potential target of antimicrobial therapy .
Iron acquisition and utilization
Iron plays a crucial role in the basic physiological functions of mammalian pathogens, e.g., ATP synthesis, formation of heme, bacterial survival and persistent infection. To overcome iron restriction in host environments, many pathogenic bacteria have evolved different approaches for iron uptake, including synthesizing certain iron-chelating compounds, called siderophores, or scavenging host iron-binding complexes . Unlike other bacterial pathogens that can produce high-affinity siderophores, such as enterobactin, alcaligin and pyochelin, it seems that H. parasuis may be deficient in production of these molecules, as reflected by the lack of related biosynthetic genes . However, H. parasuis SH0165 was found to encode at least 37 genes involved in the capture and utilization of iron from porcine transferrin, heme and heme–hemopexin (Table S6).
negative bacteria . Two clusters of consecutive genes exbB1–exbD1–tonB1 (257 aa) (HAPS1366–1364) and tonB2 (267 aa)–exbB2–exbD2 (HAPS2220–2222) were identified in the SH0165 genome, which are responsible for the formation of the TonB1 and TonB2 systems, respectively. Gene components of the TonB2 system have been described previously in H. parasuis . Although the periplasmic proteins TonB1 and TonB2 share low sequence identity (24%), they contain an identical domain (PF03544) involved in the transfer of energy to transferrin binding proteins (TbpB and TbpA), both homologues of which are responsible for transport of iron across the outer membrane of A. pleuropneumoniae . H. parasuis TbpA and TbpB (HAPS2224, 2223) have low sequence identity (28% and 42%) with H. influenzae Tbp1 and Tbp2 (HI0994, 0995), respectively, but are highly homologous to the A. pleuropneumoniae TbpA and TbpB (APJL1597, 1598) proteins, sharing 93% and 87% identity, respectively , . Consequently, the divergent residues inferred from the sequence alignments may help determine why H. parasuis and A. pleuropneumoniae preferentially bind porcine transferrin but not human transferrin .
Besides the TonB-dependent systems, H. parasuis seems to have various ABC transport systems involved in the uptake of extracellular iron, hemin and hemoprotein. A hemin transport system encoded by the hmuTUV operon (HAPS1572–1570) was identified in the SH0165 genome and was found to be homologous to a family of ABC transporters (HemTUV, ShuTUV) that function in transporting free heme into the cytoplasm . Orthologs of yfeABCD (HAPS1097–1100) and fbpABC (HAPS1129–1127) were found in A. actinomycetemcomitans and M. haemolytica, respectively, and their protein products make up periplasmic ABC transport complexes involved in iron acquisition , . In H. parasuis, the periplasmic iron transport system FbpABC together with the transferrin outer membrane receptor complex TbpAB may form another iron uptake pathway, which is used by Neisseria species to transport ferric iron into the cell .
Although the intact fhuCDBA operon involved in ferric hydroxamate uptake has been reported in strain Nagasaki of H. parasuis serovar 5 , it was found to be truncated in the SH0165 genome, only consisting of the 5′ portion of fhuC' (HAPS0095) and 3′ portion of fhuA' (HAPS0096), and lacking fhuDB. Genes hhdB' and hhdA' (HAPS1445, 1446), encoding two components of a putative hemolysin export system, and cirA' (HAPS0477), encoding an iron transporter, are also likely to be non-functional due to frameshift mutations .
In this study, we reported the complete genome sequence of H. parasuis strain SH0165, a highly virulent strain isolated in China, and further performed a comparative genomic analysis. Genomic and phylogenetic comparisons indicated that H. parasuis and A. pleuropneumoniae may derive from a recent common ancestor. Together with a full set of the relevant metabolic genes, we confirmed that H. parasuis generates ATP via both fermentation and respiration with an intact TCA cycle for anabolism. Furthermore, genes and operons related to bacterial virulence factors, such as surface polysaccharides, fimbriae and iron acquisition systems, were identified at the genomic level and compared with known virulence genes from other pathogens. A putative Wzy/Wzx-dependent biosynthetic pathway for O-chain polysaccharide was proposed and requires further experimental confirmation in H. parasuis. Our findings should provide a genetic foundation for future research into the mechanisms of pathogenesis of H. parasuis and will accelerate the development of safe and effective vaccines to prevent and control this severe swine disease.
Genes encoding enzymes involved in central metabolism of H. parasuis SH0165 and orthologs present in genomes of five representative species within Pasteurellaceae .
Genes coding for proteins related to regulatory functions of strain SH0165.
Heme biosynthetic enzymes encoded in the genome of H. parasuis SH0165 and orthologs from five representative Pasteurellaceae genomes.
Genes encoding enzymes with a role in surface polysaccharides of strain SH0165 and orthologs present in genomes of three representative Haemophilus spp.
Putative protease-encoding genes in the H. parasuis SH0165 genome.
Conceived and designed the experiments: QJ WB HC. Performed the experiments: ZX MY YF. Analyzed the data: ZX RZ. Contributed reagents/materials/analysis tools: ZX MY YF WB. Wrote the paper: ZX WB.
- 1. Biberstein EL, White DC (1969) A proposal for the establishment of two new Haemophilus species. J Med Microbiol 2: 75–78.EL BibersteinDC White1969A proposal for the establishment of two new Haemophilus species.J Med Microbiol27578
- 2. Nedbalcova K, Satran P, Jaglic Z, Ondriasova R, Kucerova Z (2006) Haemophilus parasuis and Glässer's disease in pigs: a review. Veterinarni Medicina 51: 168–179.K. NedbalcovaP. SatranZ. JaglicR. OndriasovaZ. Kucerova2006Haemophilus parasuis and Glässer's disease in pigs: a review.Veterinarni Medicina51168179
- 3. Kielstein P, Rapp-Gabrielson VJ (1992) Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J Clin Microbiol 30: 862–865.P. KielsteinVJ Rapp-Gabrielson1992Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts.J Clin Microbiol30862865
- 4. Olvera A, Calsamiglia M, Aragon V (2006) Genotypic diversity of Haemophilus parasuis field strains. Appl Environ Microbiol 72: 3984–3992.A. OlveraM. CalsamigliaV. Aragon2006Genotypic diversity of Haemophilus parasuis field strains.Appl Environ Microbiol7239843992
- 5. Oliveira S, Blackall PJ, Pijoan C (2003) Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping. Am J Vet Res 64: 435–442.S. OliveiraPJ BlackallC. Pijoan2003Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping.Am J Vet Res64435442
- 6. Oliveira S, Pijoan C (2004) Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Vet Microbiol 99: 1–12.S. OliveiraC. Pijoan2004Haemophilus parasuis: new trends on diagnosis, epidemiology and control.Vet Microbiol99112
- 7. Cai X, Chen H, Blackall PJ, Yin Z, Wang L, et al. (2005) Serological characterization of Haemophilus parasuis isolates from China. Vet Microbiol 111: 231–236.X. CaiH. ChenPJ BlackallZ. YinL. Wang2005Serological characterization of Haemophilus parasuis isolates from China.Vet Microbiol111231236
- 8. Amano H, Shibata M, Takahashi K, Sasaki Y (1997) Effects on endotoxin pathogenicity in pigs with acute septicemia of Haemophilus parasuis infection. J Vet Med Sci 59: 451–455.H. AmanoM. ShibataK. TakahashiY. Sasaki1997Effects on endotoxin pathogenicity in pigs with acute septicemia of Haemophilus parasuis infection.J Vet Med Sci59451455
- 9. del Río ML, Gutiérrez-Martín CB, Rodríguez-Barbosa JI, Navas J, Rodríguez-Ferri EF (2005) Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis. FEMS Immunol Med Microbiol 45: 75–86.ML del RíoCB Gutiérrez-MartínJI Rodríguez-BarbosaJ. NavasEF Rodríguez-Ferri2005Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis.FEMS Immunol Med Microbiol457586
- 10. Ruiz A, Oliveira S, Torremorell M, Pijoan C (2001) Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites. Clin Microbiol 39: 1757–1762.A. RuizS. OliveiraM. TorremorellC. Pijoan2001Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites.Clin Microbiol3917571762
- 11. Challacombe JF, Duncan AJ, Brettin TS, Bruce D, Chertkov O, et al. (2007) Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd. J Bacteriol 189: 1890–1898.JF ChallacombeAJ DuncanTS BrettinD. BruceO. Chertkov2007Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd.J Bacteriol18918901898
- 12. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269: 496–512.RD FleischmannMD AdamsO. WhiteRA ClaytonEF Kirkness1995Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.Science269496512
- 13. Yue M, Yang F, Yang J, Bei W, Cai X, et al. (2009) Complete genome sequence of Haemophilus parasuis SH0165. J Bacteriol 191: 1359–1360.M. YueF. YangJ. YangW. BeiX. Cai2009Complete genome sequence of Haemophilus parasuis SH0165.J Bacteriol19113591360
- 14. Salzberg SL, Delcher AL, Kasif S, White O (1998) Microbial gene identification using interpolated Markov models. Nucleic Acids Res 26: 544–548.SL SalzbergAL DelcherS. KasifO. White1998Microbial gene identification using interpolated Markov models.Nucleic Acids Res26544548
- 15. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964.TM LoweSR Eddy1997tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence.Nucleic Acids Res25955964
- 16. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35: 3100–3108.K. LagesenP. HallinEA RødlandHH StaerfeldtT. Rognes2007RNAmmer: consistent and rapid annotation of ribosomal RNA genes.Nucleic Acids Res3531003108
- 17. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.SF AltschulW. GishW. MillerEW MyersDJ Lipman1990Basic local alignment search tool.J Mol Biol215403410
- 18. Stothard P, Wishart DS (2005) Circular genome visualization and exploration using CGView. Bioinformatics 21: 537–539.P. StothardDS Wishart2005Circular genome visualization and exploration using CGView.Bioinformatics21537539
- 19. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14: 1188–1190.GE CrooksG. HonJM ChandoniaSE Brenner2004WebLogo: a sequence logo generator.Genome Res1411881190
- 20. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580.A. KroghB. LarssonG. von HeijneEL Sonnhammer2001Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J Mol Biol305567580
- 21. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.RC Edgar2004MUSCLE: multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Res3217921797
- 22. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9: 299–306.S. KumarM. NeiJ. DudleyK. Tamura2008MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences.Brief Bioinform9299306
- 23. Canchaya C, Fournous G, Chibani-Chennoufi S, Dillmann ML, Brüssow H (2003) Phage as agents of lateral gene transfer. Curr Opin Microbiol 6: 417–424.C. CanchayaG. FournousS. Chibani-ChennoufiML DillmannH. Brüssow2003Phage as agents of lateral gene transfer.Curr Opin Microbiol6417424
- 24. Sidibé M, Messier S, Larivière S, Gottschalk M, Mittal KR (1993) Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests. Can J Vet Res 57: 204–208.M. SidibéS. MessierS. LarivièreM. GottschalkKR Mittal1993Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests.Can J Vet Res57204208
- 25. Lagergård T (1995) Haemophilus ducreyi: pathogenesis and protective immunity. Trends Microbiol 3: 87–92.T. Lagergård1995Haemophilus ducreyi: pathogenesis and protective immunity.Trends Microbiol38792
- 26. Fink DL, Geme JW (2006) The Genus Haemophilus. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K.-H, Stackebrandt E, editors. The prokaryotes, 3rd ed., vol. 6. Springer, New York, NY. pp. 1034–1061.DL FinkJW Geme2006The Genus Haemophilus.M. DworkinS. FalkowE. RosenbergK.-H SchleiferE. StackebrandtThe prokaryotes, 3rd ed., vol. 6Springer, New York, NY10341061In. In.
- 27. Jahreis K, Bentler L, Bockmann J, Hans S, Meyer A, et al. (2002) Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132. J Bacteriol 184: 5307–5316.K. JahreisL. BentlerJ. BockmannS. HansA. Meyer2002Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132.J Bacteriol18453075316
- 28. Brondijk TH, Fiegen D, Richardson DJ, Cole JA (2002) Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation. Mol Microbiol 44: 245–255.TH BrondijkD. FiegenDJ RichardsonJA Cole2002Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation.Mol Microbiol44245255
- 29. Tseng CP, Albrecht J, Gunsalus RP (1996) Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli. J Bacteriol 178: 1094–1098.CP TsengJ. AlbrechtRP Gunsalus1996Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli.J Bacteriol17810941098
- 30. Perrenoud A, Sauer U (2005) Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli. J Bacteriol 187: 3171–3179.A. PerrenoudU. Sauer2005Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli.J Bacteriol18731713179
- 31. Wassarman KM (2002) Small RNAs in bacteria: diverse regulators of gene expression in response to environmental changes. Cell 109: 141–144.KM Wassarman2002Small RNAs in bacteria: diverse regulators of gene expression in response to environmental changes.Cell109141144
- 32. Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69: 183–215.AM StockVL RobinsonPN Goudreau2000Two-component signal transduction.Annu Rev Biochem69183215
- 33. Oshima T, Aiba H, Masuda Y, Kanaya S, Sugiura M, et al. (2002) Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12. Mol Microbiol 46: 281–291.T. OshimaH. AibaY. MasudaS. KanayaM. Sugiura2002Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.Mol Microbiol46281291
- 34. Buettner FF, Bendallah IM, Bosse JT, Dreckmann K, Nash JH, et al. (2008) Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence. Infect Immun 76: 2284–2295.FF BuettnerIM BendallahJT BosseK. DreckmannJH Nash2008Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence.Infect Immun7622842295
- 35. Gerlach G, Reidl J (2006) NAD+ utilization in Pasteurellaceae: simplification of a complex pathway. J Bacteriol 188: 6719–6727.G. GerlachJ. Reidl2006NAD+ utilization in Pasteurellaceae: simplification of a complex pathway.J Bacteriol18867196727
- 36. Dailey HA (2002) Terminal steps of haem biosynthesis. Biochem Soc Trans 30: 590–595.HA Dailey2002Terminal steps of haem biosynthesis.Biochem Soc Trans30590595
- 37. Dubnau D (1999) DNA uptake in bacteria. Annu Rev Microbiol 53: 217–244.D. Dubnau1999DNA uptake in bacteria.Annu Rev Microbiol53217244
- 38. Gioia J, Qin X, Jiang H, Clinkenbeard K, Lo R, et al. (2006) The genome sequence of Mannheimia haemolytica A1: insights into virulence, natural competence, and Pasteurellaceae phylogeny. J Bacteriol 188: 7257–7266.J. GioiaX. QinH. JiangK. ClinkenbeardR. Lo2006The genome sequence of Mannheimia haemolytica A1: insights into virulence, natural competence, and Pasteurellaceae phylogeny.J Bacteriol18872577266
- 39. Redfield RJ, Cameron AD, Qian Q, Hinds J, Ali TR, et al. (2005) A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. J Mol Biol 347: 735–747.RJ RedfieldAD CameronQ. QianJ. HindsTR Ali2005A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae.J Mol Biol347735747
- 40. Bigas A, Garrido ME, de Rozas AM, Badiola I, Barbé J, et al. (2005) Development of a genetic manipulation system for Haemophilus parasuis. Vet Microbiol 105: 223–228.A. BigasME GarridoAM de RozasI. BadiolaJ. Barbé2005Development of a genetic manipulation system for Haemophilus parasuis.Vet Microbiol105223228
- 41. Redfield RJ, Findlay WA, Bossé J, Kroll JS, Cameron AD, et al. (2006) Evolution of competence and DNA uptake specificity in the Pasteurellaceae. BMC Evol Biol 6: 82.RJ RedfieldWA FindlayJ. BosséJS KrollAD Cameron2006Evolution of competence and DNA uptake specificity in the Pasteurellaceae.BMC Evol Biol682
- 42. Christensen H, Kuhnert P, Olsen JE, Bisgaard M (2004) Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae. Int J Syst Evol Microbiol 54: 1601–1609.H. ChristensenP. KuhnertJE OlsenM. Bisgaard2004Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae.Int J Syst Evol Microbiol5416011609
- 43. Reid G, Sobel JD (1987) Bacterial adherence in the pathogenesis of urinary tract infection: a review. Rev Infect Dis 9: 470–487.G. ReidJD Sobel1987Bacterial adherence in the pathogenesis of urinary tract infection: a review.Rev Infect Dis9470487
- 44. Münch S, Grund S, Krüger M (1992) Fimbriae and membranes on Haemophilus parasuis. Zentralbl Veterinarmed B 39: 59–64.S. MünchS. GrundM. Krüger1992Fimbriae and membranes on Haemophilus parasuis.Zentralbl Veterinarmed B395964
- 45. Mattick JS (2002) Type IV pili and twitching motility. Annu Rev Microbiol 56: 289–314.JS Mattick2002Type IV pili and twitching motility.Annu Rev Microbiol56289314
- 46. Stevenson A, Macdonald J, Roberts M (2003) Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae. Vet Microbiol 92: 121–134.A. StevensonJ. MacdonaldM. Roberts2003Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.Vet Microbiol92121134
- 47. Watson AA, Alm RA, Mattick JS (1996) Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa. Gene 180: 49–56.AA WatsonRA AlmJS Mattick1996Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa.Gene1804956
- 48. Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744.IR HendersonF. Navarro-GarciaM. DesvauxRC FernandezD. Ala'Aldeen2004Type V protein secretion pathway: the autotransporter story.Microbiol Mol Biol Rev68692744
- 49. Pina S, Olvera A, Barceló A, Bensaid A (2009) Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains. J Bacteriol 191: 576–587.S. PinaA. OlveraA. BarcelóA. Bensaid2009Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains.J Bacteriol191576587
- 50. Olvera A, Pina S, Pérez-Simó M, Oliveira S, Bensaid A (2010) Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo. Vet Res 41: 26.A. OlveraS. PinaM. Pérez-SimóS. OliveiraA. Bensaid2010Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo.Vet Res4126
- 51. Oomen CJ, van Ulsen P, van Gelder P, Feijen M, Tommassen J, et al. (2004) Structure of the translocator domain of a bacterial autotransporter. EMBO J 23: 1257–1266.CJ OomenP. van UlsenP. van GelderM. FeijenJ. Tommassen2004Structure of the translocator domain of a bacterial autotransporter.EMBO J2312571266
- 52. Erridge C, Bennett-Guerrero E, Poxton IR (2002) Structure and function of lipopolysaccharides. Microbes Infect 4: 837–851.C. ErridgeE. Bennett-GuerreroIR Poxton2002Structure and function of lipopolysaccharides.Microbes Infect4837851
- 53. Valvano MA (2003) Export of O-specific lipopolysaccharide. Front Biosci 8: s452–471.MA Valvano2003Export of O-specific lipopolysaccharide.Front Biosci8s452471
- 54. Zucker BA, Baghian A, Traux R, O'Reilly KL, Storz J (1996) Detection of strain-specific antigenic epitopes on the lipo-oligosaccharide of Haemophilus parasuis by use of monoclonal and polyclonal antibodies. Am J Vet Res 57: 63–67.BA ZuckerA. BaghianR. TrauxKL O'ReillyJ. Storz1996Detection of strain-specific antigenic epitopes on the lipo-oligosaccharide of Haemophilus parasuis by use of monoclonal and polyclonal antibodies.Am J Vet Res576367
- 55. Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, et al. (1996) Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 4: 495–503.PR ReevesM. HobbsMA ValvanoM. SkurnikC. Whitfield1996Bacterial polysaccharide synthesis and gene nomenclature.Trends Microbiol4495503
- 56. Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, et al. (2008) Structure and genetics of Shigella O antigens. FEMS Microbiol Rev 32: 627–653.B. LiuYA KnirelL. FengAV PerepelovSN Senchenkova2008Structure and genetics of Shigella O antigens.FEMS Microbiol Rev32627653
- 57. Daniels C, Vindurampulle C, Morona R (1998) Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol Microbiol 28: 1211–1222.C. DanielsC. VindurampulleR. Morona1998Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy).Mol Microbiol2812111222
- 58. Wang L, Briggs CE, Rothemund D, Fratamico P, Luchansky JB, et al. (2001) Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes. Gene 270: 231–236.L. WangCE BriggsD. RothemundP. FratamicoJB Luchansky2001Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes.Gene270231236
- 59. Moran AP, Prendergast MM, Appelmelk BJ (1996) Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol 16: 105–115.AP MoranMM PrendergastBJ Appelmelk1996Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease.FEMS Immunol Med Microbiol16105115
- 60. Tullius MV, Munson RS Jr, Wang J, Gibson BW (1996) Purification, cloning, and expression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi. J Biol Chem 271: 15373–15380.MV TulliusRS Munson JrJ. WangBW Gibson1996Purification, cloning, and expression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi.J Biol Chem2711537315380
- 61. Gilbert M, Watson DC, Cunningham AM, Jennings MP, Young NM, et al. (1996) Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae. J Biol Chem 271: 28271–28276.M. GilbertDC WatsonAM CunninghamMP JenningsNM Young1996Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae.J Biol Chem2712827128276
- 62. Bozue JA, Tullius MV, Wang J, Gibson BW, Munson RS Jr (1999) Haemophilus ducreyi produces a novel sialyltransferase. J Biol Chem 274: 4106–4114.JA BozueMV TulliusJ. WangBW GibsonRS Munson Jr1999Haemophilus ducreyi produces a novel sialyltransferase.J Biol Chem27441064114
- 63. Shepherd JG, Wang L, Reeves PR (2000) Comparison of O-antigen gene clusters of Escherichia coli (Shigella) sonnei and Plesiomonas shigelloides O17: sonnei gained its current plasmid-borne O-antigen genes from P. shigelloides in a recent event. Infect Immun 68: 6056–6061.JG ShepherdL. WangPR Reeves2000Comparison of O-antigen gene clusters of Escherichia coli (Shigella) sonnei and Plesiomonas shigelloides O17: sonnei gained its current plasmid-borne O-antigen genes from P. shigelloides in a recent event.Infect Immun6860566061
- 64. Coyne MJ, Tzianabos AO, Mallory BC, Carey VJ, Kasper DL, et al. (2001) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis. Infect Immun 69: 4342–4350.MJ CoyneAO TzianabosBC MalloryVJ CareyDL Kasper2001Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis.Infect Immun6943424350
- 65. Bentley SD, Aanensen DM, Mavroidi A, Saunders D, Rabbinowitsch E, et al. (2006) Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet 2: e31.SD BentleyDM AanensenA. MavroidiD. SaundersE. Rabbinowitsch2006Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.PLoS Genet2e31
- 66. Chatzidaki-Livanis M, Jones MK, Wright AC (2006) Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon. J Bacteriol 188: 1987–1998.M. Chatzidaki-LivanisMK JonesAC Wright2006Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon.J Bacteriol18819871998
- 67. Zhou H, Yang B, Xu F, Chen X, Wang J, et al. (2010) Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization. Vet Microbiol 144: 377–383.H. ZhouB. YangF. XuX. ChenJ. Wang2010Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.Vet Microbiol144377383
- 68. Erbel PJ, Barr K, Gao N, Gerwig GJ, Rick PD, et al. (2003) Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli. J Bacteriol 185: 1995–2004.PJ ErbelK. BarrN. GaoGJ GerwigPD Rick2003Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli.J Bacteriol18519952004
- 69. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, et al. (1997) The complete genome sequence of Escherichia coli K-12. Science 277: 1453–1462.FR BlattnerG. Plunkett 3rdCA BlochNT PernaV. Burland1997The complete genome sequence of Escherichia coli K-12.Science27714531462
- 70. Banks KE, Fortney KR, Baker B, Billings SD, Katz BP, et al. (2008) The enterobacterial common antigen-like gene cluster of Haemophilus ducreyi contributes to virulence in humans. J Infect Dis 197: 1531–1536.KE BanksKR FortneyB. BakerSD BillingsBP Katz2008The enterobacterial common antigen-like gene cluster of Haemophilus ducreyi contributes to virulence in humans.J Infect Dis19715311536
- 71. Lichtensteiger CA, Vimr ER (2003) Purification and renaturation of membrane neuraminidase from Haemophilus parasuis. Vet Microbiol 93: 79–87.CA LichtensteigerER Vimr2003Purification and renaturation of membrane neuraminidase from Haemophilus parasuis.Vet Microbiol937987
- 72. Ingmer H, Brøndsted L (2009) Proteases in bacterial pathogenesis. Res Microbiol 160: 704–710.H. IngmerL. Brøndsted2009Proteases in bacterial pathogenesis.Res Microbiol160704710
- 73. Frase H, Lee I (2007) Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism. Biochemistry 46: 6647–6657.H. FraseI. Lee2007Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism.Biochemistry4666476657
- 74. Crosa JH, Walsh CT (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66: 223–249.JH CrosaCT Walsh2002Genetics and assembly line enzymology of siderophore biosynthesis in bacteria.Microbiol Mol Biol Rev66223249
- 75. Postle K, Kadner RJ (2003) Touch and go: tying TonB to transport. Mol Microbiol 49: 869–882.K. PostleRJ Kadner2003Touch and go: tying TonB to transport.Mol Microbiol49869882
- 76. Bossé JT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, et al. (2002) Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect 4: 225–235.JT BosséH. JansonBJ SheehanAJ BeddekAN Rycroft2002Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection.Microbes Infect4225235
- 77. Gray-Owen SD, Loosmore S, Schryvers AB (1995) Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae. Infect Immun 63: 1201–1210.SD Gray-OwenS. LoosmoreAB Schryvers1995Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae.Infect Immun6312011210
- 78. Xu Z, Zhou Y, Li L, Zhou R, Xiao S, et al. (2008) Genome biology of Actinobacillus pleuropneumoniae JL03, an isolate of serotype 3 prevalent in China. PLoS One 3: e1450.Z. XuY. ZhouL. LiR. ZhouS. Xiao2008Genome biology of Actinobacillus pleuropneumoniae JL03, an isolate of serotype 3 prevalent in China.PLoS One3e1450
- 79. Charland N, D'Silva CG, Dumont RA, Niven DF (1995) Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis. Can J Microbiol 41: 70–74.N. CharlandCG D'SilvaRA DumontDF Niven1995Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis.Can J Microbiol417074
- 80. Thompson JM, Jones HA, Perry RD (1999) Molecular characterization of the hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for hemin and hemoprotein utilization. Infect Immun 67: 3879–3892.JM ThompsonHA JonesRD Perry1999Molecular characterization of the hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for hemin and hemoprotein utilization.Infect Immun6738793892
- 81. Rhodes ER, Tomaras AP, McGillivary G, Connerly PL, Actis LA (2005) Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system. Infect Immun 73: 3758–3763.ER RhodesAP TomarasG. McGillivaryPL ConnerlyLA Actis2005Genetic and functional analyses of the Actinobacillus actinomycetemcomitans AfeABCD siderophore-independent iron acquisition system.Infect Immun7337583763
- 82. Roehrig SC, Tran HQ, Spehr V, Gunkel N, Selzer PM, et al. (2007) The response of Mannheimia haemolytica to iron limitation: implications for the acquisition of iron in the bovine lung. Vet Microbiol 121: 316–329.SC RoehrigHQ TranV. SpehrN. GunkelPM Selzer2007The response of Mannheimia haemolytica to iron limitation: implications for the acquisition of iron in the bovine lung.Vet Microbiol121316329
- 83. Faraldo-Gómez JD, Sansom MS (2003) Acquisition of siderophores in gram-negative bacteria. Nat Rev Mol Cell Biol 4: 105–116.JD Faraldo-GómezMS Sansom2003Acquisition of siderophores in gram-negative bacteria.Nat Rev Mol Cell Biol4105116
- 84. del Río ML, Navas J, Martín AJ, Gutiérrez CB, Rodríguez-Barbosa JI, et al. (2006) Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor. Vet Res 37: 49–59.ML del RíoJ. NavasAJ MartínCB GutiérrezJI Rodríguez-Barbosa2006Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor.Vet Res374959
- 85. Sack M, Baltes N (2009) Identification of novel potential virulence-associated factors in Haemophilus parasuis. Vet Microbiol 136: 382–386.M. SackN. Baltes2009Identification of novel potential virulence-associated factors in Haemophilus parasuis.Vet Microbiol136382386