Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes.
The tbpA and tbpB genes encoding the transferrin receptor proteins Tbp1 and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the Tbp1 proteins had regions of homology with Neisseria Lbp and Tbp1s and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant Tbp1 was found in inclusion bodies and could not be affinity-isolated with immobilized porcine transferrin. Most of the remaining expressed Tbp1 was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the C-lobe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbp1. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the C-lobe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.
Gonzalez
G C
GC
Department of Microbiology and Infectious Diseases, University of Calgary, AB, Canada.
Yu
R H
RH
Rosteck
P R
PR
Schryvers
A B
AB
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D015870:Gene Expression; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000737:chemistry; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014168:Transferrin / Q000378:metabolism; D033863:Transferrin-Binding Proteins
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins