Characterization of Cah, a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli.
We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Alfredo G
AG
Torres
Nicole T
NT
Perna
Valerie
V
Burland
Abdul
A
Ruknudin
Fred R
FR
Blattner
James B
JB
Kaper
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D019453:Escherichia coli O157 / Q000378:metabolism* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010641:Phenotype
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins