Actinobacillus pleuropneumoniae

The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant (aroB) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens, the HitABC proteins of Haemophilus influenzae, the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica. The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin genes.


Introduction
The low concentration of free iron, an essential nutrient for bacteria, on mucous membranes and in tissues is one of the first lines of host defense against bacterial infection. The presence of iron-binding proteins in the body fluids, such as transferrin, lactofert-in, haem, haemoglobin, and ferritin further serves to maintain low free-Fe concentrations, inhibiting bacteria growth [l]. To sequester the limited iron from the host, bacteria have evolved several mechanisms, such as the secretion of siderophores and iron chelators which compete with lactoferrin and transferrin * Corresponding author. for iron. Iron-repressible outer membrane proteins (IROMP) that serve as receptors for iron-siderophore complexes are essential for iron uptake have been identified in many pathogenic bacteria [2], ineluding Actinobacillus pleuropneumoniae [3].
A. pleuropneumoniae obtains iron from haem compounds [3] via the production hemolysins [4,5], and membrane-bound transferrin-specific receptors [6]. A. pleuropneumoniae probably binds the iron-loaded transferrin molecule to its surface and then, transports the iron from the transferrin into the cells. However, a mechanism for the transfer of iron from the transferrin to the bacterium has not been elucidated.
In this study, we reported the cloning and se- quence analysis of an iron utilization system in A.

DNA in h-dash and screening
A. pleuropneumoniae genomic DNA from different serotypes was prepared as previously described [13].
A lamda-dash library was constructed by using the genomic DNA from a serotype 5 strain as previously described [5]. A subgenomic library from serotype 5 was also constructed using BgZII and PstI digested DNA fragments separated by agarose gel electrophoresis. The 4.5-6 kbp fragments were ligated into pHG165 digested with BamHI and SaZI. The bacteriophage and subgenomic libraries were screened by hybridization using a probe (a 1033 bp PstI-XhoI DNA fragment from pYFC126) contaning the partial apxZC gene and its upstream region from A. pleuropneumoniue serotype 5 [14]. "The sequence were taken from the following sources: afuB (this study), sfua [7], fbpb [12], hitb [9] and yfuB (unpublished data

DNA sequencing and analysis
Plasmid DNA for cycle DNA sequencing was isolated with a mini-kit from Qiagen (Chatsworth, CA). The nucleotide sequence was determined by an automated fluorescence procedure based on the Sanger dideoxy chain termination method using a Taq Dye-Deoxy Terminator Cycle Sequencing kit (Applied Biosystem, Inc.). DNA sequences were determined by using double-stranded DNA templates (pNC1, pYFC178 and pJFF1052). Oligonucleotide primers based on the DNA sequence in this study were prepared by the Analytical and Synthetic Facility, Cornell University.
Both strands of the cloned DNA were completely sequenced.

Southern blotting, hybridization
A PstI and XbaI DNA fragment from pNC1 containing the afuA gene was isolated from agarose gel and labeled with [32P]dATP by nick-translation.

Results and discussion
3. I. Nucleotide sequences of the afuA, afuB and afuC genes Plasmid pNC1 and phage clone hyfc40 were se-lected from subgenomic and a-dash library, respectively. Plasmid pNC1 contains a 5.0 kb of the A. pleuropneumoniae serotype 5 chromosome, cloned into the SalI and BamHI sites of pHG165 [4,14]. hyfc40 was digested with SalI and a 9 kb fragment was ligated into pBluescriptII-SK-, to obtain pYFC178. Plasmid pJFF1052 contains a 5.8 kb chromosomal fragment of A. pleuroneumoniae serotype 1 (strain 4074) including the 5'-terminal part of the apxIC and a 5.4 kb segment upstream apxIC cloned into the XhoI site of pBluescriptII-SK- (Fig. 1). The sequences of both clones showed three open reading frames with high similarities to the s&ABC gene cluster of S. marcescens [7]. In analogy to sfuABC, the three reading frames were designated afuA, afuB, and afuC, respectively (Fig. 1). The DNA sequence of afuABC genes from A. pleuropneumoniae serotype 1 and 5 are identical.
The deduced amino acid sequence of AfuA deduced from the nucleotide sequence of afuA contains a typical signal sequence found in exported proteins. Cleavage of the signal peptide most likely occurs between the A and K residue at positions 27 and 28. AfuB is proposed to function as a cytoplasmic membrane permease and is composed of 663 amino acids, most of which are hydrophobic.
Two sequences that match the consensus permease EAA motifs (EAA---G-----------I-LP) are found (Table   Table 2 Comparison of non-polar membrane transport proteins containing sequences homologous to nucleotide-binding domains  pleuropneumoniae (this study); Hi, H. influenzae [9]; Ng, N. gonorrhoeae [12]; Sm, S. marceScens [7]; St, S. tryphimurium; Ye, Y. enterocolitica (unpublished data). N, amino-terminal half of the polypeptide. 1). These two motifs are suggested to be located on cytoplasmic loops that interact with the ATP-binding protein [16,17]. AfuC shows strong similarities to the nucleotide-binding proteins of ABC (ATP Binding Cassette) transporters [18] ( Table 2). A comparison of AfuA, AfuB and AfuC with homologous proteins are presented in Table 3. A. pleuropneumoniae together with other pathogenic bacteria possess a siderophore-independent mechanisms for iron sequestration ( [7,10,12], this study). In N. gonorrhoeae and N. meningitidis, two proteins (Tbpl and Tbp2) are responsible for binding transferrin to the cell surface [15]. Similarly, the genes for two transferrin binding proteins (Tbpl and Tbp2) have been cloned and sequenced in A. pleuropneumoniae [6]. In Neisseria species, the iron can be removed from transferrin or lactoferrin to the periplasmic space, and carried by Fbp to transport the iron molecule into the cells [15]. It has also been suggested that the iron is diffusible through the E. coli porin to the periplasm that is independent of the transferrin receptor [lo]. The mechansim of iron transport from porcine transferrin into A. pleuropneumoniae is unknown. However, the presence of afu operon homologs in H. influenzae [9,10], S. marcescens [7], and N. gonorrhoeae [ 11,121 suggests that the function of this operon may be involved in high-affinity iron acquisition from the host environment.
was studied in E. coli aroB mutant strain H1443 carrying cloned afuABC genes. As shown in Fig. 2, the afuABC genes confer the E. coli aroB mutant which is unable to synthesise enterochelin, to grow in iron-limiting medium. In medium supplemented with 0.05 mM or 0.1 mM 2,2'-dipyridyl, the strains containing the cloned ajiiABC genes or the sfuABC genes grow significantly faster and to a higher density than the non-complemented aroB mutant (Fig.   2). At 0.2 mM 2,2'-dipyridyl, growth of the aroB mutant was inhibited, but the complemented mutant was able to grow, albeit at a reduced growth rate. The growth rate of E. coli C600 (aroB) was unaffected at these concentrations, but was generally higher in this medium compared to H1443. Addition of 0.4 mM 2,2'-dipyridyl also inhibited growth of the complemented transformants of H1443, and also reduced the growth rate of the control strain E. coli C600 (aroB+). Supplementation of medium containing 0.2 mM 2.2'-dipyridyl with 2 mM Fe(SO)d restored the growth rates of H1443 and the complemented H1443 strains. However, only partial restoration of the growth rates was observed in the medium contained 0.4 mM 2,2'-dipyridyl, supplemented with Fe(S0)4. These results indicated that the importance of the functional afuABC operon for iron acquisition by complementation of the siderophore-deficient E. coli H1443 to growth on dipyridyl-containing medium.

Identification of the afuA gene by Southern blotting analysis
To examine the function of the A. pleuropneumoniae transport genes in iron uptake, iron transport "Percent similar/identical residues (S, similarity; I, identity). Percent similar residues assuming that the following amino acid pairs are equivalent; I and V, S and T, E and D, K and R, F and Y. bThe sequence were taken from the following sources: afuABC (this study), sfuABC (7), &(11,12), hit (9,lO) and yfuABC (unpublished data).
In conclusion, the afuABC operon of A. pleuropneumoniae is sufficient to enable an E. coli KlZ(aroB) mutant to grow on iron-limited medium (4 mM dipyridyl). The three polypeptides deduced from the DNA sequence were similar to that of SfuABC [7], HitABC [9,10], FbpABC [11,12] and YfuABC. Based on these data, we hypothesized that the AfuA, AfuB and AfuC polypeptides are involved the transport of ferric iron across the cytoplasmic membrane. An efficient iron acquisition system for these pathogenic bacteria may play an important role in the pathogensis of bacterial infection.

Nucleotide sequence accession number
The sequence of afuABC-apxICA genes from serotype 1 and 5 has been submitted to Genbank and assigned accession numbers UO5042 and UO4954, respectively.