Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

Abstract

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.

FIG. 1

Autoradiogram of [³⁵S]methionine-labeled proteins after incubation of C. burnetii at 42°C and pH 5.2 in a cell-free system. Samples were withdrawn after 30 (lane 2), 60 (lane 3), 90 (lane 4), 120 (lane 5), 150 (lane 6), 180 (lane 7), and 240 (lane 8) min. Lanes 1 and 9, molecular mass standards (with masses noted in kilodaltons); lane 10, protein of C. burnetii incubated at 42°C and pH 7 for 240 min. Each lane contains proteins corresponding to approximately 10⁷ bacteria.

FIG. 2

Western blots of C. burnetii proteins either prepared from bacteria isolated from continuously growing cell cultures (lanes 1 and 3) or produced by bacteria in a cell-free system at 42°C and pH 5.2 for 4 h (lanes 2 and 4). Lanes 1 and 2, anti-GroEL antiserum; lanes 3 and 4, anti-DnaK antiserum. Molecular mass standards (kDa) are marked at the left side of the Western blot.

FIG. 3

Autoradiogram of a 2-D gel with [³⁵S]methionine-labeled proteins from in vitro-induced C. burnetii (at 42°C and pH 5.2 for 4 h). The first dimension spans the pH range from 4 to 7, from left to right. The Hsp60 and Hsp70 homologues are indicated by arrows. The molecular mass standards (kDa) are marked at the left side of the autoradiogram.

FIG. 4

Alignment of the deduced amino acid sequence of the C. burnetii Hsp71 gene with four members of the Hsp70 family of proteins. Cb, C. burnetii; Lp, L. pneumophila; Ft, F. tularensis; Ec, E. coli; Cp, C. pneumoniae. Bold letters denote the N-terminal amino acid residues determined for the purified protein. Asterisks denote amino acid residues identical to residues found in the C. burnetii sequence. Shaded areas denote the three PROSITE Hsp70 family signatures.

FIG. 5

Expression of the C. burnetii 71-kDa-protein gene in E. coli. Lane 1, molecular mass standards (kDa); lane 2, clone 71; and lane 3, IPTG-induced clone 71.

FIG. 6

Immunogold staining of C. burnetii cells with primary antibodies directed to the 71-kDa protein and secondary antibodies conjugated to collodial gold. Bars represent 0.1 μm.

FIG. 7

Osborne gradient of C. burnetii membranes and (inset) Western blot of C. burnetii cell fractions with anti-DnaK antibodies. Lane 1, membrane fraction 1 (gradient fractions 1 to 5); lane 2, membrane fraction 2 (gradient fractions 19 to 22); lane 3, cytoplasmic fraction (TCA-precipitated supernatant proteins). The molecular mass standards (kDa) are marked at the left side of the Western blot.