Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction

Abstract

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.

FIG. 1.

Circular representations of the genome of B. avium. The circles represent, from the outside in: circles 1 and 2, all genes (transcribed clockwise and counterclockwise); 3, B. avium unique genes; 4, bacteriophage genes; 5, RNA genes (blue, rRNAs; red, tRNAs; and green, stable RNAs); 6, G+C content (plotted using a 10-kb window); and 7, GC deviation ([G−C]/[G+C] plotted using a 10-kb window; khaki indicates values of >1, and purple indicates values of <1). Color coding for genes is as follows: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage and insertion sequence elements; and gray, miscellaneous.

FIG. 2.

Linear genomic comparison of B. avium, B. bronchiseptica, B. pertussis, and B. parapertussis. The gray bars represent the forward and reverse DNA strands. The red and blue lines between the genomes represent protein similarity (TBLASTX) between B. avium and B. bronchiseptica or DNA-DNA similarities (BLASTN matches) between B. bronchiseptica and B. pertussis or B. parapertussis (red lines represent direct matches, while blue lines represent inverted matches).

FIG. 3.

Venn diagram showing gene complements of B. avium and B. bronchiseptica. Shown are the number of B. avium-unique CDSs (top), the number of B. bronchiseptica-unique CDSs (bottom), and the number of CDSs from one organism that have orthologues in the other (middle). Numbers in parentheses show percentages of total CDSs. Orthologous genes were identified by reciprocal best-match FASTA comparison (see Materials and Methods).

FIG. 4.

Representation of the distribution, by functional categories, of the CDSs unique to B. avium (blue), unique to B. bronchiseptica (yellow), and shared between the two organisms (purple). Figures are expressed as a percentage of the total number of CDSs in each functional category.

FIG. 5.

SDS-PAGE and Western analysis of crude LPS preparations from wild-type and mutant B. avium and B. bronchiseptica using antibodies to the band A form of LPS. Lane A, B. avium wild type; lane B, B. avium wlbA mutant; lane C, B. avium wlbL mutant; lane D, B. bronchiseptica strain RB50; and lane E, molecular mass markers (the 7-kDa band is marked).

FIG. 6.

Outer membrane protein profiles of B. avium mutants. Outer membranes isolated from iron-replete (+Fe) (36 μM FeSO₄) or iron-stressed (-Fe) (50 μM EDDHA) cultures of B. avium 4169, KO1 (the bhuR mutant), and KO1 derivatives with gene interruptions in bfrB (KO1bfrB), bfrH (KO1bfrH), and bfeA (KO1bfeA) were resolved by SDS-PAGE and stained with Coomassie brilliant blue. The two forms of BhuR (69) are denoted by arrows in the 4169 lane; asterisks (*) denote the expected locations of the polypeptides encoded by bfrB, bfrH, and bfeA. Molecular mass standards are denoted in kDa.