Characterization of the thermal stress response of Campylobacter jejuni

Abstract

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.

FIG. 1

Autoradiograph of a one-dimensional gel of proteins synthesized by C. jejuni M129. C. jejuni bacteria were preincubated at 37°C for 10 min and then either maintained at 37°C for 10 min (lane 1) or shifted to 43°C for 1, 3, or 5 min (lanes 2 to 4, respectively) to identify proteins whose synthesis is increased following a rapid temperature upshift. Proteins were labeled with [³⁵S]methionine for 15 min at either 37°C (lane 1) or 43°C (lanes 2 to 4). C. jejuni Hsps ranging in molecular mass from 28 to 93 kDa are indicated on the right (arrows). The positions of molecular mass standards (sizes are in kilodaltons) are indicated on the left.

FIG. 2

Autoradiograph of a two-dimensional gel of proteins synthesized by C. jejuni F38011. C. jejuni bacteria were preincubated at 37°C for 10 min and then either maintained at 37°C for 10 min or shifted to 46°C for 10 min to identify proteins whose synthesis is either increased or decreased following a temperature upshift. Proteins were labeled with [³⁵S]methionine for 15 min at either 37°C (A) or 46°C (B) following the temperature upshift. Fourteen proteins were identified whose synthesis was decreased following the temperature upshift (A, arrows), and 24 proteins were identified whose synthesis was increased following the temperature upshift (B, arrows). The positions of molecular mass standards (sizes are in kilodaltons) are indicated on the left. IEF, isoelectric focusing.

FIG. 3

Physical map of pMEK20. The 2,237-bp insert contains one complete ORF of 1,119 bp encoding a protein that shares similarity with DnaJ protein from other organisms and one partial ORF of 692 bp. The partial ORF represents the 3′ end of the gene that encodes the MOMP from C. jejuni. The arrows indicate the directions in which the genes are transcribed. S, Sau3AI restriction endonuclease site.

FIG. 4

Nucleotide and deduced amino acid sequences of the C. jejuni dnaJ gene. The deduced amino acid sequence is indicated below the nucleotide sequence in single-letter code. One putative ς³² (−35 [CTTGTAA] and −10 [CTTTAA]) and two putative ς⁷⁰ promoter elements are indicated by the rectangles and ovals, respectively. The proposed ribosome-binding site (AGGA) is overlined. The two in-frame translational stop codons, both of which are ochre codons, are underlined. A possible terminator is indicated by converging arrows over an inverted repeat. The circled cysteines and glycines make up the cysteine-rich repeat motif (Cys-X-X-Cys-X-Gly-X-Gly) characteristic of DnaJ proteins.

FIG. 5

Alignment of the deduced amino acid sequence of DnaJ from C. jejuni (dnaj_cj) with the DnaJ proteins from E. coli (dnaj_ecoli), S. typhimurium (dnaj_salty), and C. acetobutylicum (dnaj_cloab). Gaps, indicated by dashes, were introduced to obtain maximal alignment. Amino acids are indicated by single-letter codes and are numbered from the first valine or methionine residue. Identical or conserved amino acid residues are boxed. Comparison of the C. jejuni DnaJ amino acid sequence to the DnaJ proteins from E. coli, S. typhimurium, and C. acetobutylicum yielded amino acid sequence similarity values of 60.8, 58.9, and 59.6%, respectively.

FIG. 6

In vitro transcription-translation analysis of recombinant plasmid pMEK. Translated products were labeled with [³⁵S]methionine and separated by SDS–12.5% PAGE, and products were visualized by autoradiography. pBluescriptII SK+ (pBSKII) is the parental plasmid, and pMEK20 (dnaJ⁺) is a recombinant plasmid. The products translated by one or both of the plasmids are given on the right. The positions of molecular mass standards (sizes are in kilodaltons) are indicated on the left.