Molecular cloning and characterization of the ferric hydroxamate uptake (fhu) operon in Actinobacillus pleuropneumoniae The GenBank accession number for the sequence of the fhuCDBA operon of Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 described in this study is AF351135.

Abstract

The bacterium Actinobacillus pleuropneumoniae, a swine pathogen, utilizes ferrichrome as an iron source. This study details the molecular cloning and sequencing of the genes involved in the uptake of this hydroxamate siderophore. Four ferric hydroxamate uptake (fhu) genes, fhuC, fhuD, fhuB and fhuA, were identified in a single operon, and these were found to encode proteins homologous to proteins of the fhu systems of several bacteria, including Escherichia coli. The fhuA gene encodes the 77 kDa outer-membrane protein (OMP) FhuA, the receptor for ferrichrome. FhuD is the 356 kDa periplasmic protein responsible for the translocation of ferric hydroxamate from the outer to the inner membrane. FhuC (285 kDa) and FhuB (694 kDa) are cytoplasmic-membrane-associated proteins that are components of an ABC transporter which internalizes the ferric hydroxamate. Reference strains of A. pleuropneumoniae that represented serotypes 1 to 12 of this organism all tested positive for the four fhu genes. When A. pleuropneumoniae FhuA was affinity-tagged with hexahistidine at its amino terminus and expressed in an E. coli host, the recombinant protein reacted with an mAb against E. coli FhuA, as well as with a polyclonal pig serum raised against an A. pleuropneumoniae infection. Hence, the authors conclude that fhuA is expressed in vivo by A. pleuropneumoniae. Three-dimensional modelling of the OMP FhuA was achieved by threading it to the X-ray crystallographic structure of the homologous protein in E. coli. FhuA from A. pleuropneumoniae was found to have the same overall fold as its E. coli homologue, i.e. it possesses an N-terminal cork domain followed by a C-terminal β-barrel domain and displays 11 extracellular loops and 10 periplasmic turns.

Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor

“…The presence of fhuCDBA genes clearly supports the concept that H. parasuis strains may count for all the necessary Fhu proteins for capturing iron from siderophores. However, these results disagree with others reported recently in the literature [20] The relative position of the four genes in the H. parasuis Fhu region was identical to that of A. pleuropneumoniae [19], in which the fhuC gene is followed by the fhuD, fhuB and fhuA genes, whereas in E. coli or S. Typhimurium the fhuA is followed by fhuC, fhuD and fhuB [11,18]. The different relative position of the four genes in H. parasuis and A. pleuropneumoniae, as compared to E. coli and S. Typhimurium fhu genes, might account for the differences in the regulation of the FhuA protein expression observed in the former species.…”

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism

“…salmonis is a Gram-negative bacterium, the expressed genes also encoded proteins of the TonB system, which is a sophisticated mechanism that supplies the required energy for ferri-siderophores uptake. The TonB system includes an integral membrane protein (ExbB), a membrane-anchored periplasmatic protein (ExbD) and the TonB protein [78,79]. …”

Real-Time Quantitative Reverse Transcription-PCR Analysis of Expression Stability ofActinobacillus pleuropneumoniaeHousekeeping Genes during In Vitro Growth under Iron-Depleted Conditions

“…Furthermore, previous studies of A. pleuropneumoniae expression by polyacrylamide gel electrophoresis and immunoblotting have demonstrated that proteins encoded by tpbA and exbB are expressed only under iron-limiting growth conditions (5,25). Another A. pleuropneumoniae iron transport system, via ferrichrome, was represented in the analysis by a gene coding for ferric hydroxamate uptake protein (fhuD), which is part of the fhuCDBA operon (14). Previous results indicated that the regulation of fhuA appears to be independent of the iron supply (15).…”