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Comparative Study
. 2003 Jun;71(6):3043-52.
doi: 10.1128/IAI.71.6.3043-3052.2003.

Comparison of bipA alleles within and across Bordetella species

Affiliations
Comparative Study

Comparison of bipA alleles within and across Bordetella species

Bryna Fuchslocher et al. Infect Immun. 2003 Jun.

Abstract

The Bordetella BvgAS signal transduction system controls the expression of at least three phenotypic phases, the Bvg(+) or virulent phase, the Bvg(-) or avirulent phase, and the Bvg(i) or Bvg intermediate phase, which has been hypothesized to be important for transmission. bipA, the first identified Bvg(i)-phase gene, encodes a protein with similarity to the well-characterized bacterial adhesins intimin and invasin. Proteins encoded by the bipA genes present in Bordetella pertussis Tohama I and Bordetella bronchiseptica RB50 differ in the number of 90-amino-acid repeats which they possess and in the sequence of the C-terminal domain. To investigate the possibility that bipA alleles segregate according to host specificity and to gain insight into the role of BipA and the Bvg(i) phase in the Bordetella infectious cycle, we compared bipA alleles across members of the B. bronchiseptica cluster, which includes both human-infective (B. pertussis and B. parapertussis(hu)) and non-human-infective (B. bronchiseptica and B. parapertussis(ov)) strains. bipA genes were present in most, but not all, strains. All bipA genes present in B. bronchiseptica strains were identical to bipA of RB50 (at least with regard to the DNA sequence of the 3' C-terminal-domain-encoding region, the number of 90-amino-acid repeats encoded, and expression patterns). Although all bipA genes present in the other Bordetella strains were identical in the 3' C-terminal-domain-encoding region to bipA of B. pertussis Tohama I, they varied in the number of 90-amino-acid repeats that they encoded and in expression level. Notably, the genes present in B. parapertussis(hu) strains were pseudogenes, and the genes present in B. parapertussis(ov) strains were expressed at significantly reduced levels compared with the levels in B. pertussis and B. bronchiseptica strains. Our results indicate that there is a correlation between specific bipA alleles and specific hosts. They also support the hypothesis that both horizontal gene transfer and fine-tuning of gene expression patterns contribute to the evolution of host adaptation in lineages of the B. bronchiseptica cluster.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of the bipA loci of B. bronchiseptica RB50 and B. pertussis Tohama I. The open reading frames of B. bronchiseptica RB50 and B. pertussis Tohama I code for N-terminal signal sequences (shaded boxes), followed by ∼410-amino-acid regions with similarity to intimin and invasin (cross-hatched boxes) and then by five (B. pertussis) or eight (B. bronchiseptica) 90-amino-acid repeated domains (indicated by the numbers 1 to 5 and 1 to 8, respectively) and finally by 291-amino-acid C-terminal domains. The amino acid sequences of the C-terminal domains, as predicted from their nucleotide sequences, are shown. Restriction endonuclease sites and the locations of primers (arrows 1 to 6) used in this study are indicated. The fragment used as a probe for Southern blotting is also indicated.
FIG. 2.
FIG. 2.
Comparison of the 90-amino-acid repeat domain-containing regions of bipA within and across Bordetella species. (A) Southern blot. BsiHKI-digested genomic DNA was probed with the 870-bp KpnI-BamHI fragment shown in Fig. 1. Lane 1, B. bronchiseptica RB50; lane 2, B. parapertussisov Fr107; lane 3, B. parapertussisov JI; lane 4, B. parapertussisov C; lane 5, B. parapertussisov HI; lane 6, B. pertussis Tohama I; lane 7, B. parapertussishu 12822; lane 8, B. parapertussishu No7; lane 9, B. parapertussishu 840994; lane 10, B. parapertussishu 803; lane 11, B. parapertussishu 789; lane 12, B. pertussis 18323. The positions of molecular weight markers are indicated on the left. (B) PCR. DNA fragments were amplified from genomic DNA by using primers bBipRepF1 and bBipRepR1 (primers 3 and 4 in Fig. 1). Lane 1, B. bronchiseptica strain RB50; lane 2, B. bronchiseptica strain 590; lanes 3 to 6, B. pertussis strains Tohama I, GMT1, 6068, and CS, respectively; lane 7, B. parapertussisov Fr107; lane 8, B. pertussis strain 18323; lanes 9 to 12, B. parapertussishu strains 12822, No7, 840994, and 803, respectively. The sizes of the 1-kb-marker fragments (in kilobase pairs) are indicated on the left.
FIG. 3.
FIG. 3.
BipA protein expression as determined by Western blotting. Whole-cell lysates of B. bronchiseptica RB50 (lanes 1 to 3), B. pertussis Tohama I (lanes 4 to 6), B. pertussis 18323 (18323) (lanes 7 to 9), B. parapertussishu 12822 (lanes 10 to 12), B. parapertussishu No7 (Bvgi phase only) (lane 13), B. parapertussishu 803 (Bvgi phase only) (lane 14), B. parapertussisov Fr107 (Fr107) (lanes 15 to 17), B. parapertussisov JI (Bvgi phase only) (lane 18), and B. parapertussisov HI (Bvgi phase only) (lane 19) were analyzed by Western blotting with anti-BipA (CT4) antibody. The positions of molecular size markers (in kilodaltons) are indicated on the left. Bb, B. bronchiseptica RB50; Bp, B. pertussis Tohama I; Bp-hu, B. parapertussishu; Bp-ov, B. parapertussisov.
FIG. 4.
FIG. 4.
Comparison of the 5′ regions of bipA homologs by PCR. (A) Genomic DNA amplified by PCR by using primers KB1For and LMBipNtc. Lane 1, B. bronchiseptica RB50; lane 2, B. pertussis Tohama I; lane 3, B. parapertussisov Fr107; lane 4, B. parapertussisov JI; lane 5, B. parapertussisov HI; lane 6, B. parapertussishu 12822; lane 7, B. parapertussishu No7; lane 8, B. parapertussishu 803. (B) Genomic DNA amplified by PCR by using primers LM7BipNt and LMBipNtc. Lane 1, B. parapertussishu 12822; lane 2, B. parapertussishu No7; lane 3, B. parapertussishu 803; lane 4, B. bronchiseptica RB50; lane 5, B. pertussis Tohama I; lane 6, B. parapertussisov Fr107; lane 7, B. parapertussisov JI; lane 8, B. parapertussisov HI.
FIG. 5.
FIG. 5.
Comparison of bipA promoter regions. The nucleotide sequences of the promoter regions of B. bronchiseptica RB50, B. pertussis Tohama I, and B. parapertussisov Fr107 are shown. BvgA binding sites identified for RB50 and hypothesized to be required for transcriptional activation of bipA are indicated by arrows and are labeled IR1 and HS2 (14). BvgA binding sites hypothesized to be required for repression of bipA under Bvg+-phase conditions are also indicated by arrows and are labeled HS3, HS4, HS5, and HS6. The translation initiation codon is indicated by boldface type.
FIG. 6.
FIG. 6.
bipA expression patterns: β-galactosidase activity in B. bronchiseptica RB50 (Bb), B. pertussis GMT1 (Bp), and B. parapertussisov Fr107 (Bppov) in the Bvg+, Bvgi, and Bvg phases. The units are nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram of protein. The error bars indicate one standard deviation.
FIG. 7.
FIG. 7.
Schematic diagram of bipA alleles in strains of the B. bronchiseptica cluster. The phylogenetic relationships shown are based on the work of van der Zee et al. (58), and the dendrogram was adapted from the study of Gerlach et al. (19). The strains used in this study are included, and where known, electrophoretic types (ET) are indicated in parentheses after the strain designations. The cross-hatched area represents the region of BipA that exhibits amino acid sequence similarity with intimin and invasin, the numbered boxes represent the 90-amino-acid repeats, and the distribution of the two different C-terminal sequences are indicated (black dots on a white background, Tohama I-like; white dots on a black background, RB50-like). Although the electrophoretic types of the B. parapertussishu strains used in this study were not determined, they are almost definitely electrophoretic type 28 as all B. parapertussishu strains tested by van der Zee et al. were electrophoretic type 28 strains. The electrophoretic types of strains RB50, Cb2, and JC100 are unknown so these strains have been placed between the two large clusters of B. bronchiseptica strains. For RB50, this is consistent with its lack of IS elements. The ability to detect BipA by Western blotting (BipA exp) and the relative level of bipA expression under Bvgi-phase conditions (bipA txn) are indicated. Bppov, B. parapertussisov; Bb, B. bronchiseptica; Bpphu, B. parapertussishu; Bp, B. pertussis; UD, undetectable.

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