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1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA
2 Department of Statistics, Iowa State University, Ames, IA, USA
Correspondence
F. Chris Minion
fcminion{at}iastate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and contributes significantly to porcine respiratory disease complex (Thacker et al., 1999). It is ubiquitous throughout swine herds worldwide and is transmitted via aerosol from pig to pig. Despite aggressive vaccination programmes, M. hyopneumoniae continues to induce lesions in as many as 80 % of the pig population (Ross, 1992). It colonizes the respiratory epithelia, where it attaches exclusively to the cilia (Mebus & Underdahl, 1977), causing ciliostasis and cell death (DeBey & Ross, 1994; Livingston et al., 1972).
Like all mycoplasmas, M. hyopneumoniae must acquire essential nutrients from its host because it lacks biosynthetic pathways due to the limited coding capacity of its genome (Minion et al., 2004). One of these nutrients, iron, is an essential metal for almost all living systems. The one known exception is the agent of Lyme disease, Borrelia burgdorferi (Posey & Gherardini, 2000). Iron serves as a cofactor or as a prosthetic group for essential enzymes that are involved in basic cellular functions (O'Halloran, 1993). Free iron, however, does not exist in useful concentrations under neutral pH conditions since it is readily oxidized forming the hydroxide 
, which is soluble only at 1·4x10–9 M at pH 7 (Andrews et al., 2003). Free iron catalyses the formation of free oxygen radicals and as such is highly toxic to cells. Therefore, iron is normally bound to specific proteins in the host such as transferrin and lactoferrin to prevent its precipitation in aqueous systems and to protect the cell from damaging UV light and lipid peroxidation reactions. Transferrin, lactoferrin and lactotransferrin have iron-binding constants in the range of 
1020 (Aisen & Listowsky, 1980; Baker et al., 1994), and transferrin is never fully saturated under normal conditions, giving the host additional iron-sequestration capabilities during bacterial infections. In this way, iron is both sequestered for use by the host and use by invading pathogens is prevented as part of the innate immune defence.
Most bacteria require iron concentrations in the range of 10–6 to 10–7 M for their metabolic processes (Weinberg, 1978). Bacteria have evolved mechanisms for iron acquisition that are tightly controlled by the level and availability of iron in the environment (Ratledge & Dover, 2000), since considerable energy investment is needed by the bacteria to express competitive iron-acquisition systems (Crosa, 1989). These mechanisms involve transcriptional regulators such as Fur and cognate DNA-binding sites (Fur-binding sites) in genes involved in iron uptake (Weinberg, 1995). The function of these genes involves binding of host transferrin or lactoferrin and their reduction and uptake, siderophore production, secretion or transport systems (Ratledge & Dover, 2000). The Fur-regulated genes are also thought to contribute to virulence in many pathogens, because the existence of multiple iron-acquisition systems allows for alternative means of iron acquisition and virulence under changing environmental conditions within the host during disease. The regulation of iron-acquisition genes linked with virulence factors allows for cell invasion, and possibly cell death, increasing the iron available for uptake by the bacteria (Salyers & Whitt, 2002). Sequence analysis of the M. hyopneumoniae genome has not identified any putative fur genes or their regulators (Minion et al., 2004); it was therefore of interest to determine if M. hyopneumoniae would respond to an environment in which iron was sequestered by a chelator other than transferrin or lactoferrin.
Unlike other bacteria, mycoplasmas have minimal gene coding capacity (Hutchison & Montague, 2002) and lack cell walls, so it is thought that their interactions with the host are less complex, including the acquisition of essential nutrients. Pathogenic mycoplasmas pose interesting questions concerning iron metabolism because they lack a host-free lifestyle, they have a single membrane and few studies have been performed on iron requirements. While it has not been unequivocally shown that iron is essential for growth, Bauminger et al. (1980) showed that iron was stored in Mycoplasma capricolum and Tryon & Baseman (1987) have shown that Mycoplasma pneumoniae can take up human lactoferrin. Mycoplasmas also contain enzymes that have been shown in other systems to contain iron, such as dehydrogenases (Earhart, 1996).
Whether mycoplasmas contain complex iron-acquisition mechanisms is not known. Iron deprivation in other bacterial systems not only triggers the genes required for its acquisition but also in many pathogens is a signal for controlling virulence factor expression (Lamont et al., 2002; Rodriguez & Smith, 2003; Schaible & Kaufmann, 2004; Szczebara et al., 1999). Thus, virulence factors can sometimes be identified by their response to low-iron growth conditions. In these studies, 2,2'-dipyridyl was used to chelate iron in growing M. hyopneumoniae cultures, and experiments using microarrays showed that iron deprivation in vitro leads to transcriptional changes. Both up- and down-regulated genes were identified.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cultures were passed fewer than 15 times in vitro in Friis media as previously described (Friis, 1975). Twelve 250 ml flasks containing 125 ml cultures were grown at 37 °C to early exponential phase as determined by medium colour change and optical density. To six flasks, 2,2'-dipyridyl (Sigma) was added to a final concentration of 1 mg ml–1. Six flasks were left untreated, and all flasks were incubated for 2 h at 37 °C. Cells were pelleted by centrifugation at 24 000 g, and 500 µl RNAlater (Ambion) was added to the pellet. Pellets were stored at –70 °C until the total RNA was isolated.
To assess the effect that various iron chelators would have on mycoplasma growth, Mycoplasma gallisepticum (ATCC 19610) and Mycoplasma pulmonis UAB6510 (Davis et al., 1980) were grown in modified Hayflick's medium as described except that the medium was supplemented with 25 µg ml–1 cefoperazone (Pfizer) instead of thallium acetate and benzylpenicillin (Freundt, 1983). Hayflick's medium was supplemented with 2,2'-dipyridyl (0·2 and 1 mg ml–1), sodium citrate (1, 2, 5 and 7·5 mM) and deferoxamine mesylate (desferal) (0·2 and 1 mg ml–1), and growth was determined by plating 10-fold serial dilutions of these broth cultures at different time points onto Hayflick's agar medium lacking chelators. Colony-forming units (c.f.u.) were determined after incubation at 37 °C for 4–5 days.
Microarray.
The M. hyopneumoniae microarray consists of PCR products (probes) spotted to Corning UltraGAPS glass substrates. Ninety-one per cent (632/698) of the M. hyopneumoniae ORFs are represented on the array as PCR products 125–350 bp in length. Each product is a unique sequence even within paralogous families as described by Minion et al. (2004). The absence of 66 ORFs is due to an inability to design suitable primers (12 ORFs) or failed PCR reactions due to incorrect product size, multiple bands or no band (54 ORFs). Only ORFs larger than 125 bp are represented on the array and no tRNAs or rRNAs were included. The primer design, array construction and validation have been described (Madsen et al., 2006).
Experimental design.
Six independent RNA samples from iron-depleted cells were paired with six independent RNA samples from control cells for hybridization on six two-colour microarrays. For three of the six arrays, the control sample was labelled with Alexa 555 dye and compared to the iron-depleted sample labelled with Alexa 647 dye (Molecular Probes). The dye assignment to control and treated samples was reversed for the other three arrays. The three slides were hybridized under identical conditions as described below.
RNA isolation.
RNA was isolated from frozen cell pellets using the Versagene RNA Purification System (Gentra Systems) according to the manufacturer's protocol. The optional step of DNase treatment was performed on column according to the manufacturer's recommendation. With a cut-off of 150 bp, 5S rRNA and tRNAs were removed from the samples, limiting interference in downstream manipulations. Samples were quantified and checked for purity using a Nanodrop ND-1000 spectrophotometer. If necessary, samples were concentrated using Microcon YM-30 micro-concentrators (Millipore) for optimal cDNA generation.
Target generation and hybridization.
Fluorescently labelled cDNA targets were generated and purified using the SuperScript Indirect cDNA Labelling System (Invitrogen) with a set of 129 ORF-specific hexamer oligonucleotide primers designed as previously described (Madsen et al., 2006). Following purification of the labelled cDNA, samples were dried in a vacuum centrifuge and then resuspended in 10 µl Pronto! cDNA/long oligo hybridization solution (Corning). Targets were denatured at 95 °C for 5 min and centrifuged at 13 000 r.p.m. for 2 min at room temperature. Targets from a control and an iron-deprived culture were then combined, pipetted onto an array, and covered with a 22x22 mm HybriSlip (Schleicher & Schuell). Slides were placed in a Corning hybridization chamber and incubated in a 42 °C water bath for 12–16 h. Slides were washed according to Corning's UltraGAPS protocol and dried by centrifugation.
Data acquisition and normalization.
Each array was scanned with each dye channel using a ScanArray Express laser scanner (Applied BioSystems) three times under varying laser power and photomultiplier tube gain settings to increase the dynamic range of expression measurement (Dudley et al., 2002). Images were quantified using the softWorRx Tracker analysis software package (Applied Precision). Spot-specific mean signals were corrected for local background by subtracting spot-specific median background intensities. The natural logarithms of the background-corrected signals from a single scan were adjusted by an additive constant so that all scans of the same array-by-dye combination would have a common median. The median of these adjusted-log-background-corrected signals across multiple scans was then computed for each spot to obtain one value for each combination of spot, array and dye channel. These data for the two dye channels on any given array were normalized using locally weighted scatterplot smoother (LOWESS) normalization to adjust for intensity-dependent dye bias (Dudoit et al., 2000; Yang et al., 2002). Following LOWESS adjustment, the data from each channel were adjusted by an additive constant so that the median for any combination of array and dye would be the same for all array-by-dye combinations. The normalized values for triplicate spots were averaged within each array to produce one normalized measure of expression for each of the 632 probe sequences and each of the 12 RNA samples.
Data analysis.
A separate mixed linear model analysis was conducted for each probe sequence using the normalized data (Wolfinger et al., 2001). Each mixed model included fixed effects for treatment (iron depletion versus control), slide region (upper versus lower) and dye (Alexa 555 versus 647) as well as random effects for slide and slide-by-region interaction. A t-test for differential expression across treatments was conducted for each probe as part of our mixed linear model analyses. The 632 P-values from these t-tests were converted to q-values using the method of Storey & Tibshirani (2003). These q-values can be used to obtain approximate control of the false discovery rate at a specified value. For example, declaring probes with q-values less than or equal to 0·05 to be differentially expressed produces a list of significant results for which the false discovery rate is estimated to be approximately 5 %. Along with q-values, estimates of fold change were computed for each probe by taking the inverse natural log of the mean treatment difference estimated as part of our mixed linear model analyses.
Confirmation of microarray data.
To confirm significant transcriptional differences between genes, semi-quantitative RT-PCR analysis was performed on a subset of genes that showed significant up- or down-regulation in the microarray analysis. The PCR was performed in 96-well plates in an MJ Research Dyad Thermal Cycler (Bio-Rad) in 20 µl reactions. The six cDNA preparations for each control and iron-depleted sample were used with previously described conditions (Madsen et al., 2006). The reactions were analysed by agarose gel electrophoresis, stained and digitized, and each band density was measured with FluorChem 8000 version 2.0 software (Alpha Innotech). Analysis of variance was performed to determine differences between band density values of iron-depleted vs control samples. Band densities were background corrected by subtracting the background density within the reaction lane from the sample band density. Estimated differences were considered significant if the P-value of a t-test was <0·05.
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RESULTS |
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shows the results of supplementing Hayflick's growth medium with 2,2'-dipyridyl at 0·2 and 1 mg ml–1. This demonstrates that 2,2'-dipyridyl consistently slowed growth but only at the highest concentration (1 mg ml–1). At no time was growth completely stopped. Neither sodium citrate nor desferal had an effect on growth at any concentration tested (data not shown). Assuming that 2,2'-dipyridyl has the same effect on M. hyopneumoniae growth, subsequent iron-limiting studies with M. hyopneumoniae were performed using a concentration of 1 mg ml–1.
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Data from each of the six replicates were used in the statistical analysis. Statistical analysis indicated that 27 genes had significant transcriptional differences with a P-value <0·01. The results are presented in Table 1 and Fig. 2. Nine genes demonstrated a significant increase in transcriptional activity under iron-limiting conditions compared to control cells grown under normal in vitro conditions. Eighteen genes, however, were down-regulated under these conditions. Fig. 2 displays the results of the 632 genes as a volcano plot, with up- and down-regulated genes marked. The data for selected genes are shown in Fig. 3. These data are shown as normalized data, and each line in the graph connects the two conditions, iron depletion and control, for one array. The data for all six arrays used in the statistical analysis are plotted. The quality of the data is reflected by the similarity in the slopes of the lines for each gene.
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. A 16S RNA control reaction was included. RT-PCR reactions were performed using the 12 RNA samples from the control and low-iron studies. The reactions contained equal RNA concentrations and were performed at the same time to reduce experimental error. Reaction products were separated on an agarose gel, stained, and the band intensities quantified. The twelve experimental replicates for each gene (six control and six low-iron samples) were analysed for significance. The RT-PCR product signal intensities for genes mhp140, mhp151 and mhp558 from low-iron conditions were significantly different from the control at P<0·01 (Fig. 4). Genes mhp275 and mhp639 were not significantly different at P=0·01.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). M. genitalium did not bind either protein. Iron storage occurs in M. capricolum (Bauminger et al., 1980), but iron acquisition or iron requirements have not been studied further in the mycoplasmas.
The rationale for using chelators in this study was to expose the mycoplasma to conditions that prevent iron uptake through normal pathways. This should be reflected in slower or lack of growth under the iron-limiting conditions, assuming there is a requirement for iron. Our initial studies used M. gallisepticum and M. pulmonis with three different iron chelators to estimate the need for iron among the mycoplasmas and to identify a chelator and concentration that might provide a slower growth response. We examined a range of chelator concentrations from 0·1 to 1 mg ml–1; the lower limit reflects concentrations used in other bacterial systems (Delany et al., 2001; Paustian et al., 2001; Rosner et al., 2002) and the upper limit was thought necessary to provide sufficient chelator for serum-supplemented medium. Since both species demonstrated reduced growth with 2,2'-dipyridyl at 1 mg ml–1 (Fig. 1
), this chelator and concentration were used in transcriptional profiling studies with M. hyopneumoniae. We also chose a single time point, 2 h, because it represents an approximate generation time for M. hyopneumoniae under the growth conditions employed.
Analysis of the microarray data indicates that nine genes were up-regulated during iron deprivation: iolB (mhp151), iolC (mhp152), glpQ (mhp317), mglA (mhp319), ackA (mhp505) and potB (mhp558), in addition to three hypothetical genes (mhp140, mhp275 and mhp510). One of the latter, mhp275, is a member of the P102 paralogue family (Minion et al., 2004). The function of P102 or any of its paralogues is not known, but it is interesting to speculate that one of these might have a role in iron uptake. Some of the known genes (mglA and potB) are involved in transport, while others (iolCB, iolC, glpQ and ackA) are involved in metabolism. Interestingly, mhp319 (mglA) is the only member of the ATP transporter family (there are 13 homologues in the genome) that showed differential transcription during iron deprivation. Could this be a transporter involved in iron uptake? Possibly, but without additional information, this would be entirely speculative. One of the genes, mhp140, has a prokaryotic membrane lipid attachment site and is thought to be a lipoprotein (Minion et al., 2004).
Eighteen genes were down-regulated (P<0·01) during iron limiting conditions. Seven of these (mhp081, mhp087, mhp176, mhp337, mhp411, mhp639 and mhp666) are hypothetical genes with no identified function. The remaining genes are identified as gatB (mhp030), fusA (mhp083), rpsL (mhp085), hpt (mhp117), oppB–D (mhp167–169), rpL2 (mhp190), metK (mhp450), rplL (mhp637) and ushA (mhp651). Resistance to fusidic acid in fusA mutants of Salmonella enterica serovar Typhimurium alters growth both in vivo and in vitro (Macvanin et al., 2003). So, although the serovar Typhimurium fusA mutants have some antibiotic resistance, their ability to grow is also stunted. The down-regulated genes identified in this study have roles in translation and cell growth. The gene products of oppB–D are involved in oligopeptide uptake, and as such, their down-regulation fits well with the idea of a slowed metabolism. The lack of biosynthetic pathways makes amino acid uptake a requirement in mycoplasmas. Alternatively, down-regulation of oppB–D might indicate a need for the mycoplasma to replace normal activities of the membrane with iron-acquisition components, although this is entirely speculative. The regulation or role of ushA during transcription is not clearly defined; however, it is known to increase twofold as cells enter the stationary phase (Wanner, 1996). In M. hyopneumoniae this protein has a prokaryotic lipid attachment site and is thought to be a lipoprotein. The down-regulation of ushA is most likely a response to iron depletion and not due to cells entering the stationary growth phase under our experimental conditions. hpt is involved in the salvage of guanine for guanine nucleotide synthesis (Zalkin & Nygaard, 1996). Since cellular metabolism and growth are slowed during iron deprivation, the need to synthesize nucleotides is expected to decrease as well.
The degree of transcriptional change that occurs during low-iron growth conditions may indicate that iron-acquisition systems in mycoplasmas are tightly controlled. The mechanism(s) employed to accomplish this remain unknown. The number of hypothetical genes affected by iron chelation in this study was consistent with the 44 % of hypothetical genes in the genome (10 of 27). Some of these may play important roles in M. hyopneumoniae's response to iron stress. Since there is no clear indication of the functions of these genes, additional studies are required to determine their role in iron acquisition and/or cellular metabolism. When comparing the iron deprivation data with our previous heat shock data (Madsen et al., 2006), three of the nine up-regulated genes, mhp151, mhp152 and mhp275, are up-regulated under both conditions (P<0·01). For those genes down-regulated during iron deprivation, three are also down-regulated during heat shock (P<0·01) (mhp030, mhp176 and mhp337). This suggests that these six genes might be considered ‘stress response’ genes.
The evaluation of samples taken from the lungs of swine at the height of infection may be informative in conjunction with the data found here. However, laboratory-grown organisms may be competing for iron in vastly different ways than those found in their host niche. How M. hyopneumoniae secures sufficient levels of iron for its metabolism and whether it is able to respond to low-iron conditions through transcriptional regulation is not yet understood, but studies on the regulation mechanism(s) of several genes identified during this study may begin to unravel some of the mystery.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Andrews, S. C., Robinson, A. K. & Rodriquez-Quinones, F. (2003). Bacterial iron homeostasis. FEMS Microbiol Rev 27, 215–237.[CrossRef][Medline]
Baker, E. N., Anderson, B. F., Baker, H. M., Day, C. L., Haridas, M., Norris, G. E., Rumball, S. V., Smith, C. A. & Thomas, D. H. (1994). Three-dimensional structure of lactoferrin in various functional states. Adv Exp Med Biol 357, 1–12.[Medline]
Bauminger, E. R., Cohen, S. G., Labenski-Dekanter, F., Levy, A., Aster, S., Kessel, M. & Rottem, S. (1980). Iron storage in Mycoplasma capricolum. J Bacteriol 141, 378–381.
Crosa, J. H. (1989). Genetics and molecular biology of siderophore-mediated iron transport in bacteria. Microbiol Rev 53, 517–530.
Davis, J. K., Delozier, K. M., Asa, D. K., Minion, F. C. & Cassell, G. H. (1980). Interactions between murine alveolar macrophages and Mycoplasma pulmonis in vitro. Infect Immun 29, 590–599.
DeBey, M. C. & Ross, R. F. (1994). Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun 62, 5312–5318.
Delany, I., Spohn, G., Rappuoli, R. & Scarlato, V. (2001). The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol Microbiol 42, 1297–1309.[CrossRef][Medline]
Dudley, A. M., Aach, J., Steffen, M. A. & Church, G. M. (2002). Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range. Proc Natl Acad Sci U S A 99, 7554–7559.
Dudoit, S., Yang, Y. H., Callow, M. J. & Speed, T. P. (2000). Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. http://www.stat.berkeley.edu/users/terry/zarray/Html/papersindex.html
Earhart, C. F. (1996). Uptake and metabolism of iron and molybdenum. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 1075–1090. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Freundt, E. A. (1983). Culture media for classic mycoplasmas. In Methods in Mycoplasmology, vol. 1, Mycoplasma Characterization, pp. 127–135. Edited by S. Razin & J. G. Tully. New York: Academic Press.
Friis, N. F. (1975). Some recommendations concerning primary isolation of Mycoplasma hyopneumoniae and Mycoplasma flocculare, a survey. Nord Veterinaermed 27, 337–339.
Hutchison, C. A., III & Montague, M. G. (2002). Mycoplasmas and the minimal genome concept. In Molecular Biology and Pathogenicity of Mycoplasmas, pp. 221–253. Edited by S. Razin & H. Herrmann. New York: Kluwer Academic/Plenum.
Lamont, I. L., Beare, P. A., Ochsner, U., Vasil, A. I. & Vasil, M. L. (2002). Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 99, 7072–7077.
Livingston, C. W., Stair, E. L., Underdahl, N. R. & Mebus, C. A. (1972). Pathogenesis of mycoplasmal pneumonia in swine. Am J Vet Res 33, 2249–2258.[Medline]
Macvanin, M., Bjorkman, J., Eriksson, S., Rhen, M., Andersson, D. I. & Hughes, D. (2003). Fusidic acid-resistant mutants of Salmonella enterica serovar Typhimurium with low fitness in vivo are defective in RpoS induction. Antimicrob Agents Chemother 47, 3743–3749.
Madsen, M. L., Nettleton, D., Thacker, E. L., Edwards, R. & Minion, F. C. (2006). Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays. Infect Immun 74, 167–174.
Mare, C. J. & Switzer, W. P. (1965). New species: Mycoplasma hyopneumoniae, a causative agent of virus pig pneumonia. Vet Med Small Anim Clin 60, 841–846.[Medline]
Mebus, C. A. & Underdahl, N. R. (1977). Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae. Am J Vet Res 43, 1249–1254.
Minion, F. C., Lefkowitz, E. L., Madsen, M. L., Cleary, B. J., Swartzell, S. M. & Mahairas, G. G. (2004). The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186, 7123–7133.
O'Halloran, T. V. (1993). Transition metals in control of gene expression. Science 261, 715–725.
Paustian, M. L., May, B. J. & Kapur, V. (2001). Pasteurella multocida gene expression in response to iron limitation. Infect Immun 69, 4109–4115.
Posey, J. E. & Gherardini, F. C. (2000). Lack of a role for iron in the Lyme disease pathogen. Science 288, 1651–1653.
Ratledge, C. & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54, 881–941.[CrossRef][Medline]
Rodriguez, G. M. & Smith, I. (2003). Mechanisms of iron regulation in mycobacteria: role in physiology and virulence. Mol Microbiol 47, 1485–1494.[CrossRef][Medline]
Rosner, J. L., Dangi, B., Gronenborn, A. M. & Martin, R. G. (2002). Posttranscriptional activation of the transcriptional activator Rob by dipyridyl in Escherichia coli. J Bacteriol 184, 1407–1416.
Ross, R. F. (1992). Mycoplasmal disease. In Diseases of Swine, pp. 537–551. Edited by A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire & D. J. Taylor. Ames: Iowa State University Press.
Ross, R. F. (1993). Mycoplasma-animal pathogens. In Rapid Diagnosis of Mycoplasmas, pp. 69–109. Edited by I. Kahane & A. Adoni. New York: Plenum.
Salyers, A. A. & Whitt, D. D. (2002). Bacterial Pathogeneis: a Molecular Approach, 2nd edn. Washington, DC: American Society for Microbiology.
Schaible, U. E. & Kaufmann, S. H. E. (2004). Iron and microbial infection. Nat Rev Microbiol 2, 946–953.[CrossRef][Medline]
Storey, J. D. & Tibshirani, R. (2003). Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100, 9440–9445.
Szczebara, F., Dhaenens, L., Armand, S. & Husson, M. O. (1999). Regulation of the transcription of genes encoding different virulence factors in Helicobacter pylori by free iron. FEMS Microbiol Lett 175, 165–170.[CrossRef][Medline]
Thacker, E. L., Halbur, P. G., Ross, R. F., Thanawongnuwech, R. & Thacker, B. J. (1999). Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J Clin Microbiol 37, 620–627.
Tryon, V. V. & Baseman, J. B. (1987). The acquisition of human lactoferrin by Mycoplasma pneumoniae. Microbial Pathog 3, 437–443.
Wanner, B. L. (1996). Phosphorus assimilation and contol of the phosphate regulon. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 1362–1381. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Weinberg, E. D. (1978). Iron and Infection. Microbiol Rev 42, 45–66.
Weinberg, E. D. (1995). Acquisition of iron and other nutrients in vivo. In Virulence Mechanisms of Bacterial Pathogens, pp. 79–93. Edited by J. A. Roth and others. Washington, DC: American Society for Microbiology.
Wolfinger, R. D., Gibson, G., Wolfinger, E. D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C. & Paules, R. S. (2001). Assessing gene significance from cDNA microarray expression data via mixed models. J Comput Biol 8, 625–637.[CrossRef][Medline]
Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. & Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method for addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.
Zalkin, H. & Nygaard, P. (1996). Biosynthesis of purine nucleotides Washington, DC: American Society for Microbiology.
Received 8 November 2005;
revised 5 January 2006;
accepted 11 January 2006.
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), 0·2 mg ml–1 (
) and 1 mg ml–1 (
) 2,2'-dipyridyl and incubated at 37 °C for the indicated time. Samples were removed, serially diluted and plated on mycoplasma agar lacking chelator for c.f.u. counts at the times indicated. Data represent the means of triplicate platings.
