Characterization of pit, a Streptococcus pneumoniae iron uptake ABC transporter

Abstract

Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus pneumoniae operon, named pit, encoding proteins with similarity to components of a putative Brachyspira hyodysenteriae iron uptake ABC transporter, Bit. An S. pneumoniae strain containing a defined mutation in pit has impaired growth in medium containing the iron chelator ethylenediamine di-o-hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of S. pneumoniae systemic infection. Furthermore, addition of a mutation in pit to a strain containing mutations in the two previously described S. pneumoniae iron uptake ABC transporters, piu and pia, resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual pit, piu, and pia mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that pia is the dominant iron transporter during in vitro and in vivo growth.

FIG. 1.

(A) Genetic organization of the pit locus. Thick black line, chromosomal DNA; open boxes, pit ORFs (A, putative iron-binding lipoprotein receptor gene; B and C, putative transmembrane genes; D, putative ATPase gene); arrows, site of insertion in mutant strains. (B) Transcriptional analysis of the pit locus. Ethidium bromide-stained agarose gels containing products with the same primer pairs for PCR with S. pneumoniae chromosomal DNA as a template (on the left) and RT-PCR with S. pneumoniae RNA as the template (on the right) are shown in subpanel ii. RT-PCR mixtures containing no reverse transcriptase generated no products. Bars marked in subpanel i represent the corresponding target products for each pair of primers used. Boxes in subpanel iii show the results of Southern hybridizations of the corresponding PCR products when probed with a product representing the whole pit locus.

FIG. 2.

Cladogram showing the relationship of PitA, PiuA, and PiaA to each other and their close homologs in the finished and unfinished bacterial genomes (marked by their name if published or accession or annotated genome number if not). unf., unfinished genome data; Ap, Actinobacillus pleuropneumoniae; Bd, B. hyodysenteriae; Bs, Bacillus subtilis; Cc, Campylobacter coli; Cj, Campylobacter jejuni; Cd, C. diphtheriae; Ef, Enterococcus faecalis; Ec, E. coli; Hi, Haemophilus influenzae; Pm, Pasteurella multocida; Se, S. equi; Sa, S. aureus; Sm, Sinorhizobium meliloti; Smu, Streptococcus mutans; Spy, S. pyogenes; Va, Vibrio anguillarum; Yp, Y. pestis.

FIG. 3.

(A and B) Growth curves as measured by OD of the pitA mutant (circles) and wild-type (diamonds) strains in THY (A) and in THY plus 200 μM EDDA (B). (C) Growth curves as measured by OD of the piuB/piaA (squares) and the piuB/piaA/pitA (triangles) mutant strains in THY (open symbols), Chelex-THY (black symbols), and Chelex-THY-50 μm FeCl₃ (grey symbols). Results for panels A to C are representative curves from experiments performed three times. (D) Maximum OD during growth in RPMIm broth for pitA mutant strains and strains containing double mutations in two of the three genes piuB, piaA, and pitA compared to the wild-type strain. Results are the means of three separate samples, and the bars represent SDs. For the difference in maximum OD between the piuB/piaA and the piuB/piaA/pitA mutant strains, P was <0.001.

FIG. 4.

Sensitivity to streptonigrin of the pitA mutant strains. Results are expressed as the radius of growth inhibition (millimeters) surrounding an antibiotic disk impregnated with streptonigrin. (A) Comparison of the results with 5-μg streptonigrin disks for the piuB, piaA, and pitA single mutant strains cultured on RPMIm plates and on RPMIm plates containing 400 μM DIP. For the pitA mutant strain versus the wild-type strain, P was <0.001 (on RPMIm) and 0.001 (on RPMIm plus DIP); for the piaA mutant strain versus the piuB or pitA mutant strain, P was <0.05 (on RPMIm) and <0.001 (on RPMIm plus DIP). (B) Comparison of the results with 20-μg streptonigrin disks for the piuB/piaA/pitA triple mutant strain and the piuB/piaA double mutant strain cultured on RPMIm plates and on RPMIm plates containing 400 μM DIP. For the piuB/piaA mutant strain versus the piuB/piaA/pitA mutant strain, P was 0.09 (on RPMIm) and <0.001 (on RPMIm plus DIP). Error bars represent the SDs.

FIG. 5.

Comparison of the ⁵⁵FeCl₃ content after 30 min of incubation for the wild-type, pitA mutant, piaA/pitA and piuB/piaA double mutant, and piuB/piaA/pitA triple mutant strains. Mean data from three assays per strain are presented and normalized for bacterial CFU. For the differences between the wild-type strain and the pitA mutant strain, P was 0.9; for the differences between the wild-type strain and the piaA/pitA, piuB/piaA, and the piuB/piaA/pitA mutant strains, P was ≤0.001; for the differences between the piuB/piaA mutant strain and the piuB/piaA/pitA mutant strain, P was 0.02. Error bars represent the SDs.

FIG. 6.

Relative abundance of piu, pia, and pit mRNA transcripts. (A) Efficiency of PCRs for piuB (lanes 1), piaA (lanes 2), and pitA (lanes 3) with DNA as the template. Product quantity was assessed by running 8 μl of product after 16, 20, and 24 cycles on a 1.5% agarose gel and staining the product with ethidium bromide. (B) PCRs for piuB, piaA, and pitA with cDNA made from RNA obtained from wild-type bacteria grown in Chelex-THY. Product quantity was assessed by running 8 μl of product after 16, 20, 24, and 28 cycles on a 1.5% agarose gel and staining the product with ethidium bromide. The kilobase ladder is presented in the lane marked kb.