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J Bacteriol. Feb 2008; 190(4): 1495–1496.
Published online Dec 7, 2007. doi:  10.1128/JB.01845-07
PMCID: PMC2238223

The Complete Genome Sequence of Actinobacillus pleuropneumoniae L20 (Serotype 5b)[down-pointing small open triangle]

Abstract

There are 16 capsule-based serotypes of Actinobacillus pleuropneumoniae, all of which are capable of causing disease in pigs. Here we report the finished and annotated genome sequence of the reference serotype 5b strain L20. This strain has a rough appearance and readily forms biofilms, as is typical for most field isolates (6).

The complete genome sequence of Actinobacillus pleuropneumoniae L20 was determined at the Natural Research Council of Canada, using a shotgun sequencing strategy. The shotgun library consisted of 25,000 clones containing 1.5- to 2.0-kb inserts. Draft assemblies were compiled using Consed (4), based on 36,267 reads, to give 7.6-fold coverage of the genome. Gaps between contigs were closed by direct sequencing, with chromosomal DNA as the template.

The A. pleuropneumoniae L20 genome is 2,274,482 bp in length and contains 2,012 putative open reading frames (ORFs), which were predicted using Glimmer (11). Preliminary annotation of identified ORFs was achieved on the basis of BLAST and Smith-Waterman searches against different databases. Manual curation was performed in order to avoid annotation drift.

No plasmid DNA was found in L20, although plasmids have been described for other strains of this species. The chromosome of L20 does not contain duplicate regions. The average GC content is 41.3%, similar to those of other members of the Pasteurellaceae (9). The genes encoding the serotype 5b-specific capsule, i.e., cps5C, cps5B, and APL_1581 (a putative glycosyltransferase gene), have significantly lower GC contents (average of 28.2% over the 3,745 bp comprising the three genes) than those of the surrounding genes (kdsA, 41.7%; and cpxD, 44.1%), indicating recent acquisition of these capsule genes. Two ORFs (APL_1465 and APL_1466) within a cluster of lipopolysaccharide-related genes also have a low GC content (27.1% over both genes) compared to those of the surrounding genes (wzm, 35.4%; and rfbD, 39%). Although there are a few isolated genes encoding hypothetical proteins that have GC contents of <30%, there are no other extended regions of significant GC disparity. Even the flp-tad cluster, which shows complete synteny with a 14-gene island encoding the bundle-forming Flp pili, involved in biofilm formation in Aggregatibacter (Actinobacillus) actinomycetemcomitans and other bacteria (7), has an overall GC content of 37.7%. This island shows greater divergence in GC content than does the genome in A. actinomycetemcomitans and in Pasteurella multocida, whereas the degree of divergence in Haemophilus ducreyi is more similar to that in A. pleuropneumoniae (7).

Approximately 86% of the nucleotides are predicted to be involved in coding. There are 16 pseudogenes, including hsdM, prrC, and lpsA, all of which have internal stop codons. The genome contains a number of transposons, including nine instances of IS3 family-like (IS150 group) transposons, two instances of IS1595 family-like transposons, and one instance of an IS481 family-like transposon, although none are inserted in known ORFs.

The genome contains 62 tRNA and 7 rRNA operons. Genes encoding the RNA polymerase subunits (rpoA, rpoB, rpoC, and rpoZ) are present, as are five genes encoding different sigma factors. In addition to rpoD, rpoH, rpoN, and rpoE, there is a gene (APL_1831) encoding another RpoE-like extracellular function (ECF) sigma factor. We are currently studying the extracytoplasmic stress response regulon of A. pleuropneumoniae, which is controlled by RpoE. Unlike other ECF sigma factor genes, APL_1831 is not cotranscribed with a gene encoding a cognate anti-sigma-factor protein (8), and the function of this putative sigma factor is not known. There is no rpoS homologue in the A. pleuropneumoniae L20 genome. We have identified five predicted two-component regulatory systems, encoded by arcA/arcB, cpxA/cpxR, narQ/narP, phoB/phoR, and qseB/qseC.

Some members of the Pasteurellaceae, most notably Haemophilus influenzae and Haemophilus somnus, are able to variably express certain virulence-associated genes via slipped-strand base mispairing involving short repetitive DNA sequences (5, 12, 14). The repeat units can range from monomeric to octameric tracts (3, 12) and can be located either within ORFs, where slipped-strand base mispairing results in interruption or size modification of the ORF, or in promoter regions, where alteration of spacing of binding domains determines binding of the RNA polymerase (12, 13). The presence of tandem repeat sequences may be indicative of the contribution of gene products to virulence (5). We analyzed the A. pleuropneumoniae L20 genome for the presence of tandem DNA repeats by using EMBOSS (10) and found three potential phase-variable genes. In the L20 genome, there are 18 tandem copies of a 6-bp repeat (CAGCTC) present at the 3′ end of hlp, which encodes a putative lipoprotein with 18 Pro-Ala repeats in the carboxy terminus. The serotype 1 strain 4074 hlp gene has one less copy, resulting in the loss of one of the Pro-Ala repeats. Immediately upstream of a gene encoding a predicted adenine-specific modification enzyme (APL_0705), there are 14 tandem copies of GCACA. In the serotype 1 4074 sequence, there are only 13 tandem copies of this repeat, which are present immediately following the initiation codon of this gene. The extra repeat in the serotype 5b L20 sequence results in a frame shift that introduces a stop codon such that the gene is transcribed from a subsequent initiation codon. The L20 adenine-specific modification enzyme is 30 amino acids shorter than the strain 4074 protein. The third tandem repeat sequence, TAGTGA, is present in five copies in the middle of a hypothetical gene that in serotype 1 4074 (GenBank accession number ZP_00348288) has only four copies of this repeat. The translation of the L20 sequence has five Ser-Asp repeats, whereas the 4074 protein has four Ser-Asp repeats.

We previously reported that the genome of A. pleuropneumoniae L20 contains 742 copies of the 9-bp sequence ACAAGCGGT, a DNA uptake signal sequence (USS) that has also been identified in Mannheimia haemolytica and H. ducreyii but which differs from the USS found in H. influenzae and other members of the Pasteurellaceae (9). The genome has a full complement of known competence genes (9). This is consistent with L20 being naturally transformable via a USS-dependent mechanism (1).

The genome sequence of A. pleuropneumoniae L20 has been used in the construction of a DNA microarray (for details on the construction and content of the AppChip1 microarray, see the Institute for Biological Sciences website [http://ibs-isb.nrc-cnrc.gc.ca/glycobiology/appchips_e.html]), providing a valuable tool for transcriptional profiling (2) as well as for genomotyping studies.

Nucleotide sequence accession number.

The complete genome sequence of the A. pleuropneumoniae strain L20 has been assigned GenBank accession number CP000569.

Acknowledgments

Funding for the sequencing project was provided by the National Research Council Genomics and Health Initiative (phases 1 and 2). J.B. was supported by a BBSRC grant (to P.L.).

We thank Rama Singh for managing the shotgun sequencing, Sharen Bowman for managing the closing stages, and Sonia Leclerc for other sequencing reactions.

Footnotes

[down-pointing small open triangle]Published ahead of print on 7 December 2007.

REFERENCES

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