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The necessity to sequester essential biochemical processes poses a challenge for living cells. Physical compartmentalization created by semipermeable membrane partitions achieves this segregation, but at the predictable expense of imposing a restrictive barrier to the cellular ingress and exit of solutes. Prokaryotes and eukaryotes have surmounted this obstacle by evolving transport systems, termed ATP-binding cassette (ABC) transporters, that couple ATP hydrolysis with substrate translocation across biological membranes (2, 6, 12, 24). These systems exhibit a modular organization comprised of four structural domains that may be expressed as individual polypeptides or may be fused into single multidomain proteins. Two membrane-integral domains span the membrane multiple times and form the passageway through which the solute flux occurs. Two ATP-binding cassettes reside on the cytosolic face of the membrane.

The binding-protein-dependent transporters of gram-negative bacteria represent the best-characterized members of this superfamily. These transporters recruit an auxiliary component, a periplasmic binding protein, that constitutes the major determinant in conferring substrate specificity (2, 6, 7, 12, 20, 26). The genes encoding this transporter complex are arranged as a single transcriptional unit, although apparent exceptions exist in which the periplasmic binding protein genes are unlinked from their cognate permease genes (8, 15, 23).

In the pathogenic neisseria Neisseria gonorrhoeae and Neisseria meningitidis, a gene cluster, termed fbpABC, has been postulated to mediate the delivery of iron across the periplasmic space into the cytoplasm (1, 5). The iron acquisition phenotype of a meningococcal fbpABC mutant supports this proposal for the obligatory participation of fbpABC in neisserial periplasmic iron transport from human transferrin and human lactoferrin (14). This locus displays the signature core components characteristic of binding-protein-dependent ABC transporters. Biochemical studies (5) identify FbpA as the substrate binding protein. The functional assignments of FbpB as the cytoplasmic membrane protein and of FbpC as the ATPase subunit are implied by the deduced amino acid sequence similarity to homologous proteins (1).

It remains unclear whether the genes in this locus are cotranscribed as a single expression unit. Nucleotide sequence (1) and primer extension (9) analyses have mapped a single potential promoter site upstream of fbpA. However, Northern blot hybridization did not disclose the presence of a polycistronic transcript (9). Therefore, this investigation was undertaken to address the proposed operonic organization of fbpABC by conducting transcription assays that exploit the sensitivity of reverse transcriptase (RT)-PCR amplification.

Bacterial strains and growth conditions. The bacteria used in this study are listed in Table 1. Neisserial strains were grown on chocolate agar at 35°C in an atmosphere of 5% CO₂. A single colony was selected from overnight growth on chocolate agar to inoculate 10 ml of brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) containing a 50 µM concentration of the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenylacetic acid)]. The culture was grown in a shaking incubator at 37°C in the presence of 5% CO₂ until mid-logarithmic growth was achieved (equivalent to an optical density at 600 nm [OD₆₀₀] of 0.685 as measured with a Pye Unicam PU8800 spectrophotometer).

DNA isolation and manipulations. Meningococcal genomic DNA was recovered by standard methods (21). DNA fragments were purified from agarose gels by using either the GeneClean II Purification Matrix kit (BIO 101, Inc., Vista, Calif.) or by passage through NENSORB 20 (NEN, Boston, Mass.) cartridges. DNA sequencing was performed according to the dideoxynucleotide chain-termination method (22) with a PRISM Ready Reaction dye cycle sequencing kit (Applied Biosystems) with fluorescence-labelled synthetic oligonucleotide primers based on the known fbpABC sequence (1). All sequence reactions were run and analyzed on an Applied Biosystems 377XL automated DNA sequencer.

RNA isolation and RT-PCR. Total cellular RNA was extracted from mid-logarithmic-phase (OD₆₀₀ of 0.685) meningococcal cultures by using the RNeasy Midi kit (Qiagen, Inc., Clarita, Calif.) according to the manufacturer's recommendations. To eliminate contaminating genomic DNA, total RNA was subjected to DNase I (amplification grade, Gibco BRL, Life Technologies, Burlington, Canada) treatment as specified by the manufacturer. RNA concentrations were determined by measuring the A₂₆₀; samples were immediately stored at 70°C. Reverse transcription was performed with the SuperScript II RNase H RT-PCR kit (Gibco BRL) following the manufacturer's instructions. The indicated gene-specific primers (Table 1) initiated first-strand cDNA synthesis. Thirty-six cycles of PCR amplification were performed with Taq polymerase (Gibco BRL) on a Perkin-Elmer model 480 DNA thermal cycler with denaturation at 94°C for 30 s, primer annealing at 52°C for 30 s, and extension at 72°C for 3 min. Identical aliquots were processed in parallel without the addition of RT, in order to ensure that residual genomic DNA was not serving as the template in the PCR amplification. PCR amplification products were electrophoresed on 1% agarose gels and stained with ethidium bromide. The identity of all RT-PCR amplification fragments was verified by nucleotide sequencing.

QRT-PCR. For quantitative RT-PCR (QRT-PCR), cDNA synthesis was performed in a 20-µl final volume that included 2 µg of meningococcal total RNA, 100 pmol of random hexamer oligonucleotides (N6) as primers, RT buffer (50 mM Tris [pH 8.3], 75 mM KCl, 1.5 mM MgCl₂), 10 mM dithiothreitol, and 1 mM (each) deoxynucleoside triphosphates (dNTPs; dATP, dGTP, dCTP, and dTTP), 100 U of Superscript II RT (Gibco BRL), and 17 U of RNase inhibitor (RNAguard; Amersham Pharmacia Biotech, Inc., Baie d'Urfé, Canada). The RT reaction was performed in an MJ Research minicycler PTC-150 at 22°C for 5 min, followed by incubation at 4°C for 50 min. The samples were heated for 5 min at 95°C to terminate the reaction. Real-time quantitative PCR was performed in 10-µl final volumes in glass capillaries in a LightCycler Instrument (Roche Diagnostics, Laval, Canada) (29). The PCR master mix comprised 1× PCR buffer, 3 mM MgCl₂, 1 mg of bovine serum albumin per ml, 0.2 mM dNTPs, 0.5 µM both forward and reverse primers, a 1:3,000 dilution of SYBR Green I (Molecular Probes), and 0.4 U of Platinum Taq (Gibco BRL). Into each capillary tube, 9 µl of PCR master mix and 1 µl of template target DNA (cDNA or pCR2FABC) were loaded. Sealed capillaries were centrifuged prior to placement into the LightCycler carousel. PCR amplification was performed with an initial denaturation at 95°C for 30 s followed by 45 cycles of denaturation at 95°C for 2 s (ramping at 20°C/s), annealing at 52°C for 5 s (ramping at 20°C/s), and elongation at 72°C for 42 s (ramping at 5°C/s). Amplicon specificity was verified by melting curve analyses with the LightCycler software, version 3.39. The identity of the amplicons was also established by confirmation of the expected molecular weight by agarose gel electrophoresis. Optimal conditions for amplification were determined by preliminary experiments with meningococcal genomic DNA. The quantitative PCR experiment was repeated three times, and each experiment produced similar results.

Transcription assays using RT-PCR. The integrity of the total RNA preparation was assessed by demonstrating the presence of the transcript from the housekeeping gene asd (10) (Fig. 1B, lane h; gene-specific primer 3'HKasdstop for the RT step, primer pair 5'HKasdint and 3'HKasdstop for PCR amplification) and the presence of the iron-regulated transcript tbpA (Fig. 1B, lane d; gene-specific oligonucleotide 48 for the RT step, primers 385 and 48 for the PCR step). The latter result also suggests that the starting RNA preparation is unlikely to be selectively biased against iron-regulated transcripts. The requirement for such a representative mRNA library arises from two considerations. First, the transcription of fbpA is enhanced under iron-limiting conditions (9). Second, given the proposed operonic organization of fbpABC, the expression of the putative polycistronic transcript encompassing this gene cluster would be anticipated to exhibit the same property.

View larger version (53K): [in this window] [in a new window]   FIG. 1.   (A) The meningococcal fbpABC gene cluster encoding the neisserial iron ABC transporter. The orientations and approximate positions of the gene-specific primers for generating the cDNA and the oligonucleotide primers used in the PCR amplification step of the RT-PCR assays are displayed below the schematic. gDNA, genomic DNA. (B) RT-PCR amplification of total RNA extracted from N. meningitidis B16B6. The PCR amplification products were fractionated in a 1% agarose gel that was stained with ethidium bromide. The ingredients used to generate the amplicon in each lane are depicted in the table above the agarose gel. RT primers are designated as follows: tbp, 48; Asd, 3'HKasdstop; B, 3'fbpBint; and C, 3'fbpCstop. PCR primer pairs are designated as follows: tbpA, 385 and 48; asd, 5'HKasdint and 3'HKasdstop; AB, 5'fbpAint-3'fbpBint; and BC, 5'fbpBint-3'fbpCint. The figure was imaged with a Hewlett-Packard ScanJet HP, edited by using Adobe Photoshop 3.0, and labelled by using Microsoft PowerPoint 97. The sizes of standards in kilobases (lane a) are shown on the left.

Transcript quantity. Real-time PCR studies were used to determine the relative abundance of fbpA-, fbpAB-, and fbpBC-bearing transcripts.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   (A) Kinetic PCR curves for primer pair 560 and 561. Relative fluorescence output is plotted versus PCR cycle number. Kinetic curves are for 107 (A), 106 (B), 105 (C), and 103 (D) pCR2FABC DNA template copy numbers and for random-hexamer-primed cDNA (E). (B) Calibration curve for primer pair 560 and 561. The Ct value from each kinetic curve is plotted versus the log of the initial DNA concentration. (C) Copy number measured by real-time QRT-PCR. The copy number of standard pCR2FABC DNA added to the reactions is calculated from the moles of standard pCR2FABC added multiplied by Avogadro's number (6 × 1023). Given the Ct of each sample, the initial copy number is calculated from the calibration curve conducted during the same experiment. Each assay was performed in triplicate, and representative data from one such experiment are shown. The figure was imaged with a Hewlett-Packard ScanJet HP, edited by using Adobe Photoshop 3.0, and labelled by using Microsoft PowerPoint 97.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   (A) Kinetic PCR curves for primer pair 5'fbpAint and 3'fbpBint. Relative fluorescence output is plotted versus PCR cycle number. Kinetic curves are for 106 (A), 105 (B), 104 (C), and 103 (D) pCR2FABC DNA template copy numbers and for random hexamer-primed cDNA (E). (B) Calibration curve for primer pair 3'fbpBint. The Ct value from each kinetic curve is plotted versus the log of the initial DNA concentration. (C) Copy number measured by real-time QRT-PCR. The copy number of standard pCR2FABC DNA added to the reactions is calculated from moles of standard pCR2FABC added multiplied by Avogadro's number (6 × 1023). Given the Ct of each sample, the initial copy number is calculated from the calibration curve conducted during the same experiment. Each assay was performed in triplicate, and representative data from one such experiment are shown. The figure was imaged with a Hewlett-Packard ScanJet HP, edited by using Adobe Photoshop 3.0, and labelled by using Microsoft PowerPoint 97.