Characterization of a periplasmic ATP-binding cassette iron import system of Brachyspira (Serpulina) hyodysenteriae

Abstract

The nucleotide sequence of the pathogenic spirochete Brachyspira hyodysenteriae bit (for "Brachyspira iron transport") genomic region has been determined. The bit region is likely to encode an iron ATP-binding cassette transport system with some homology to those encountered in gram-negative bacteria. Six open reading frames oriented in the same direction and physically linked have been identified. This system possesses a protein containing ATP-binding motifs (BitD), two hydrophobic cytoplasmic membrane permeases (BitE and BitF), and at least three lipoproteins (BitA, BitB, and BitC) with homology to iron periplasmic binding proteins. These periplasmic binding proteins exhibit lipoprotein features. They are labeled by [(3)H]palmitate when tested in recombinant Escherichia coli, and their signal peptides are typical for substrates of the type II secretory peptidase. The FURTA system and Congo red assay indicate that BitB and BitC are involved in iron binding. The Bit system is detected only in B. hyodysenteriae and is absent from B. innocens and B. pilosicoli.

FIG. 1

Restriction map of the Bit A′BCDEF system and the different subclones. The pDJ1 insert was subcloned from the SacI sites of phage λGEM-11 clone 42 into the pBluescriptII KS plasmid. The pDJ2 plasmid corresponds to the SacI-ClaI fragment of pDJ1. pDJ3, pDJ4, pDJ5, and pDJ6 correspond, respectively, to the ClaI, PstI, EcoRV, and BglII restriction fragments subcloned from pDJ1. pDJ7 and pDJ8 correspond, respectively, to the SacI-EcoRV and SacI-BglII fragments of pDJ1 ligated in pBluescriptII KS. Finally, pDJ9 corresponds to the BglII-BglII fragment of pDJ1. The parenthetical sizes refer to the length of the cloned DNA fragment and do not include the vector length. The arrows indicate the directions and lengths of the ORFs. The Western blot results with anti-B. hyodysenteriae serum are shown on the right. Restriction sites: B, BglII; C, ClaI; E, EcoRV; H, HindIII; N, NdeI; Ns, NsiI; P, PstI, S, StuI. In FURTA, + indicates that LacZ from fhuF::lacZ is expressed and − indicates that LacZ is not expressed. In Congo red binding (CRB), + indicates that the colonies are red on Trypticase soy agar plates containing Congo red and − indicates that the Congo red is not bound by the strains (white). The asterisk indicates that the immunoreactive protein is composed of the first 296 amino acids of BitB and the last 46 amino acids of BitC.

FIG. 2

Western blot analysis. Expression of recombinant proteins of pDJ plasmids as determined by Western blot analysis using a rabbit antiserum directed against B. hyodysenteriae serotype 8. Lanes: 1, B. hyodysenteriae serotype 8; 2, pDJ1; 3, pDJ2; 4, pDJ3; 5, pDJ4; 6, pDJ5; 7, pDJ6; 8, pDJ7; 9, pDJ8; 10, detection of Bit-responsive proteins in B. hyodysenteriae with an antiserum against the recombinant BitB-expressing E. coli.

FIG. 3

Possible evolutionary tree for the periplasmic binding proteins (PBP). Homologous sequences were aligned with Clustal, and the relationship between sequences were calculated by the unweighted pair group method with arithmetic mean with the GeneWorks program (IntelliGenetics, Inc.).

FIG. 4

[³H]palmitate labeling of BitB and BitC. Lanes: 1, whole-protein content of recombinant E. coli XL1 Blue containing the pBluescriptII KS plasmid; 2 and 3, pDJ5 and pDJ2 transformants, respectively, immunoprecipitated as described in Materials and Methods. Between 5 and 10 μg of proteins was added to each well.

FIG. 5

Electron micrograph of negatively stained B. hyodysenteriae (A) and B. innocens (B) cells incubated with anti-Bit antibodies and protein A-colloidal gold.

FIG. 6

Hybridization of BglII fragments of genomic DNAs by using probe 42 derived from the ClaI-SacI fragment of pDJ2. Lanes: 1, B. hyodysenteriae serotype 8 ATCC 49887; 2, E. coli HB101; 3, B. innocens; 4, B. hyodysenteriae B78.