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Previous Article | Next Article 
Journal of Bacteriology, June 2007, p. 3996-4013, Vol. 189, No. 11
0021-9193/07/$08.00+0 doi:10.1128/JB.00185-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
sodA1 Mutant to Paraquat Reveal Metal Ion Homeostasis Imbalances during Endogenous Superoxide Stress
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Department of Microbiology and Immunology,1 Bioinformatics Program, University of Michigan Medical School,2 Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 481093
Received 4 February 2007/ Accepted 12 March 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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sodA1) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the sodA1 mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and sodA1 cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The sodA1 mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in B. anthracis. A double mutant lacking both the sodA1 and sodA2 genes (sodA1 sodA2) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between sodA1 and sodA2, and a role for the sod genes in manganese homeostasis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Changes in the intracellular and extracellular redox environment can affect cells by altering or damaging biomolecules (20). Reactive metabolites such as hydrogen peroxide and superoxide are normal intracellular by-products of aerobic metabolism, and conserved molecular mechanisms such as the scavenging enzymes catalase and superoxide dismutase (SOD) are present in aerobic organisms for protection from these endogenously produced oxidatively active species (18, 42, 46). Because these metabolites are unique in their chemical properties, cellular transcriptional responses to them are often distinctive. Additionally, reactive molecules such as superoxide and nitric oxide are ubiquitous signaling molecules in eukaryotic systems (3, 28, 29). During the establishment and progression of anthrax, reactive oxygen metabolites may be encountered by B. anthracis exogenously, such as from the oxidative burst of phagocytic cells of the innate immune system (19, 35, 59) and in the extracellular milieu established in certain tissues of a host (34, 47, 75). Since superoxide is a charged radical, it is not diffusible across membranes, and the exogenous encounter with this reactive oxygen metabolite presumably would not affect a transcriptional program (24). However, when bacteria are growing rapidly and to very high titers within the host during late infection, they are presumably generating metabolic by-products of their own (63) and, depending on the mode of central metabolism, will need to adjust the transcriptional program to support optimal growth.
In this study, we used custom Bacillus anthracis Affymetrix GeneChips (4) to define and compare the global transcriptional responses of B. anthracis Sterne (34F2) and an isogenic mutant lacking the SOD gene sodA1 (sodA1) to endogenous superoxide stress induced by the superoxide-generating compound paraquat. The sodA1 gene has recently been shown to be necessary for protection from this type of oxidative stress (62), and the comparison of the responses of Sterne and the sodA1 mutant after this major physiological redox perturbation identified a transcriptional program involving genes for metal ion transport, siderophore biosynthesis, putative glyoxalase enzymes, and oxidoreductase activity. The substantial increase in the transcription of the genes involved in the biosynthesis of the iron-chelating siderophore bacillibactin after paraquat treatment led us to quantify extracellular and intracellular levels of both of the siderophores produced by B. anthracis (bacillibactin and petrobactin) in order to determine how siderophore production is influenced by oxidative stress. Because the transcriptional program after endogenous superoxide stress suggests a metal ion imbalance, the growth of the sodA1 and sodA2 mutants and of the sodA1 sodA2 double mutant in various types of metal-limiting media was also explored.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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sod, asb, and bac mutants has been described previously (45, 62) All were constructed using the Sterne 34F2 parental strain. Spores of all strains were prepared in modified G medium as described previously (62). When antibiotics were used, concentrations for B. anthracis strains were 30 µg/ml for kanamycin and 5 µg/ml for erythromycin.
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sodA1 strain were diluted 1:100 into fresh LB medium in the morning and allowed to recover for approximately 2 h. Cells were back-diluted to an optical density at 600 nm(OD600) of 0.01 in an 80-ml volume of fresh LB medium. Cells were allowed to grow to an OD600 of 0.4 to 0.5. The cultures were split into three separate 20-ml cultures, and methyl viologen (paraquat) (Aldrich 856177) was added to two of the flasks to a final concentration of 100 µM or 800 µM. The third flask was left untreated as a control. Cultures were allowed to grow for approximately 20 to 25 min. A 6-ml volume of cells was collected from each culture and pelleted by centrifugation at 4°C for 7 min at 
2,000 rpm. RNA was isolated using the Ambion RiboPure-Bacteria kit according to the manufacturer's instructions with the following modifications: cell disruption with zirconia beads was done for 15 min, 400 µl of RNAwiz reagent was used, BCP phase separation reagent was used in place of chloroform, and 50 µl of RNase- and DNase-free distilled water was added during extraction to optimize the yield. The QIAGEN RNeasy minikit with an on-column DNase digestion step was used according to the manufacturer's RNA cleanup protocol. RNA was quantified via the A260:A280 ratio on a Beckman DU530 spectrophotometer. RNA integrity was checked using an Agilent 2100 bioanalyzer at the University of Michigan Affymetrix cDNA core facility. The procedures described above were carried out in three independent experiments utilizing unique spore starter cultures each time. Microarray sample processing and data collection. RNA samples were reverse transcribed, and the corresponding cDNA samples were purified, fragmented, and labeled according to Affymetrix-recommended protocols (available at http://www.affymetrix.com/support/downloads/manuals/expression_s3_manual.pdf) at the University of Michigan Comprehensive Cancer Center microarray core facility. Hybridization to the B. anthracis GeneChips (4), as well as scanning of the arrays, was also done according to standard Affymetrix protocols. At this point, several quality control steps were performed in order to ensure that the raw data were of sufficient quality to proceed. First, the distributions of perfect-match probe intensities for each chip were compared, since the robust-multichip-average procedure used for normalization and background correction over multiple chips is based on the assumption that these distributions are very similar. Then, a plot of average probe intensity versus position within a gene was generated for each sample. This plot shows if there are systematic skews within a given data set toward probes that lie near the end of each gene, which would indicate a high level of RNA degradation or problems with the reverse transcription (RT) step. Once it was verified that all the samples showed similar 5'-to-3' profiles, the robust-multichip-average method was used to subtract background, normalize data, and compute a single probe set summary for each gene (8, 43, 44). Principal-component analysis verified that biological replicates were very similar to each other and formed relatively tight clusters (data not shown).
Microarray data analysis. Significance analysis of microarrays (SAM) was performed using the TMeV 3.1 (version 10.2) suite of programs (http://www.tm4.org/) (68a). Note that in all cases, SAM were done with a false discovery rate of <0.001. Pathway analysis of array data was done using the EASE algorithm (39) as implemented within the TM3-MeV program, as well as a set of GO and TIGRFAM tables compiled from the TIGR Comprehensive Microbial Resource (http://www.tigr.org/CMR/). The significance of overrepresentations was assessed using the Fisher exact text. Note that only those genes that showed statistically significant up- or down-regulation and at least a twofold change in expression are listed in the tables and in the supplemental material.
Assays of growth under iron- and manganese-limiting conditions.
Growth curves assessing the growth of B. anthracis strains in media containing the iron chelator 2,2'-dipyridyl and in media predepleted of cations were performed as follows. For chelated medium, LB medium alone or with 300 µM or 400 µM 2,2'-dipyridyl was dispensed in 100-µl volumes into flat-bottom, 96-well tissue culture-treated polystyrene plates (Corning-Costar 3628). The vegetative bacilli of the strains indicated were grown to mid-exponential phase (OD600, 0.4 to 0.5), washed three times in sterile metal-depleted medium, and then back-diluted to an OD600 of approximately 0.08 to 0.10. Culture growth was assessed in a Molecular Devices Spectramax M2 spectrophotometer for 10 to 12 h at 37°C; A600 readings were taken every 10 min, with shaking for 560 s (9.3 min) between readings. For depleted medium, experiments were performed as for chelated medium but with iron- and manganese-depleted medium (IMnDM), with or without addition of Fe(II)SO4·7H2O or MnSO4·H2O to a final concentration of 20 µM each. IMnDM was prepared like iron-depleted medium (IDM) (as reported previously [13]) but without the addition of manganese salts. Cations were depleted by using Bio-Rad Chelex 100.
Doubling times were calculated as follows, according to the method of Moat et al. (56). First, the variable n was calculated as [log10(high OD600) – log10(low OD600)]/0.3010, where OD600 values are from exponentially dividing cells. Then the doubling time was calculated as [time (in minutes) between the high OD600 and the low OD600]/n.
The MIC of 2,2'-dipyridyl for spore outgrowth was determined as follows. LB medium with 0, 100, 200, 300, 400, and 500 µM 2,2'-dipyridyl was prepared and distributed in 100-µl volumes in flat-bottom 96-well tissue culture-treated polystyrene plates (Corning-Costar 3628). Approximately 105 spores of each strain were added to each well (three separate strain stocks in three separate experiments). Outgrowth was assessed as the ability to establish exponential growth to an OD600 of at least 0.3 after 8 h of incubation at 37°C in a Molecular Devices Spectramax M2 spectrophotometer, with A600 readings taken every 10 min and with shaking for 560 s (9.3 min) between readings.
End point RT-PCR.
A total of 700 ng of RNA collected from B. anthracis Sterne (34F2) and the sodA1 mutant (as described above) was used to perform end point RT-PCR using Invitrogen one-step RT-PCR with Platinum Taq according to the manufacturer's instructions. Briefly, RT was performed at 50°C for 30 min. PCR was performed with 0.25 pg of operon- or gene-specific primers (sequences available upon request) for 37 cycles with an elongation temperature of 70°C and an extension time of 30 s. Five microliters of each PCR product was run on a 0.7% Tris-borate-EDTA agarose gel and visualized by ethidium bromide staining. Negative controls omitting reverse transcriptase and positive controls with B. anthracis Sterne (34F2) and sodA1 genomic DNAs were run with each experiment and primer pair. Operon- or gene specific primers were designed to result in products of approximately 200 bp.
SYBR green quantitative RT-PCR.
RNA was collected from Sterne and sodA1 cells as described above. A total of 700 ng of RNA was used to make cDNA using 300 ng of random primers and Invitrogen SuperScript II reverse transcriptase, by following the maker's protocol, with an overnight incubation at 42°C. For all samples, a control without reverse transcriptase was performed. Diagnostic PCR of the cDNA was done using 1.5 µl of each reaction product to eliminate the possibility of genomic DNA contamination (all results were negative) and for the confirmation of DNA synthesis using primers for the fusA gene.
SYBR green PCR was performed on an ABI Prism 7900HT system at the University of Michigan cDNA Affymetrix core facility at the Comprehensive Cancer Center. The Applied Biosystems SYBR green master mix was used with 1.5 µl of the cDNA preparations. Each reaction was performed twice with two biological replicates, for a total of four reactions per gene-specific primer pair per condition (with cDNA, without cDNA, and without reverse transcriptase). Forty elongation cycles were performed with 30-s elongation times and an annealing temperature of 55°C (optimized beforehand), with a melting temperature of 94°C and an extension temperature of 68°C.
Gene-specific primers used corresponded to overlapping segments of the first two genes in the bac operon (GBAA2369 and -2370) and internal segments of the following genes: the second gene in the asb operon (GBAA1982), the gene encoding the glyoxalase family protein (GBAA3339), the ccdA-1 cytochrome c-type biogenesis protein gene (GBAA1778), the thioredoxin family protein gene (GBAA1779), and the prolipoprotein diacylglyceryl transferase family protein gene (GBAA1780). (All PCR products were between 180 and 200 bp. Primer sequences are available upon request).
Threshold cycle (CT) values are the values where the PCR enters exponential amplification. Approximate transcript abundance was analyzed using the comparative CT method (or 
method). The CT for the fusA control PCR product is subtracted from each of the experimental CTs. The difference in transcript abundance is then calculated as 2–(difference in normalized CTs). CTs used are averages for four reactions.
Quantification of the siderophores bacillibactin and petrobactin.
Three-milliliter overnight cultures of B. anthracis Sterne 34F2 and the sodA1 and bac mutants were added to 100 ml of LB medium or IDM (13) in 500-ml plastic flasks. At an OD600 of approximately 0.4 to 0.5, paraquat (methyl viologen) was added to a final concentration of 800 µM. Cultures were incubated for 1 h at 37°C with shaking at 300 rpm. Supernatants were collected via filtration with Corning 0.2-µm-pore-size vacuum filter flasks.
For cell lysates, cells from filter flasks were resuspended in 20 ml of sterile phosphate-buffered saline, pH 7.4 (without calcium chloride or magnesium chloride). Cells were freeze-thawed three times, once at –20°C and twice at –80°C. Cell suspensions were sonicated six times with 30-s pulses at 4°C. Cell lysates were pelleted by centrifugation, and lysate supernatants were incubated on ice with 18 µl of DNase (RNase free) (180 U) for 1 h. Lysates were then spun by centrifugation at 9,000 x g for 35 min, and lysate supernatants were collected for analysis. Total protein concentrations were assayed using the Qubit protein assay (Molecular Probes).
For quantification of bacillibactin, cytosolic fractions (5 ml) from LB or IDM cultures were adjusted to pH 2.0 and then extracted three times with equal volumes of ethyl acetate. The organic layers were pooled, and the solvents were concentrated into dryness in vacuo. The dried pellets were dissolved in 50 µl of methanol and used for quantitative analysis of intracellular bacillibactin. Samples were prepared for quantification of extracellular bacillibactin by extracting acidified (pH 2.0) LB or IDM culture supernatants (10 ml) with an equal volume of ethyl acetate three times. Organic fractions were pooled and concentrated into 100 µl of methanol for analysis.
For quantification of petrobactin, cytosolic fractions (5 ml) or supernatants (10 ml) from LB or IDM cultures were adjusted to pH 7.0. XAD-16 resin was added (1 g for the cytosolic fraction, 2 g for supernatants), and the mixtures were shaken for 1 h at 150 rpm in the dark. Mixtures were then filtered, the resin was washed with pure water three times, and extracts were eluted with methanol (5 ml for the cytosolic fraction and 10 ml for supernatants). Methanol eluates were concentrated to dryness in vacuo and dissolved in 50 µl (cytosolic) or 100 µl (supernatant) of 80% methanol for quantification.
The organic solvent or XAD-16 resin extracts from the culture supernatants or cytosolic fractions of B. anthracis were analyzed using a Shimadzu LCMS-2010EV system. (The liquid chromatography-mass spectrometry [LC-MS] analysis is described in detail in the next section.) 2,3-Dihydroxybenzoic acid (for bacillibactin) and 3,4-dihydroxybenzoic acid (for petrobactin) were utilized as internal standards for signal normalization. Ten microliters of a 2,3-dihydroxybenzoic acid or 3,4-dihydroxybenzoic acid stock solution (1 mM) was added to 100 µl of each sample. By the LC methods described below, bacillibactin and 2,3-dihydroxybenzoic acid showed retention times of 19.230 to 19.340 and 17.367 min, and petrobactin and 3,4-dihydroxybenzoic acid showed retention times of 15.860 to 16.134 and 18.133 min, respectively. The calibration curves were constructed by using a series of 10 concentrations (ranging from 10 to 500 µM) of authentic bacillibactin and petrobactin for quantification. The retention time and mass spectrum of the corresponding bacillibactin or petrobactin peak in each sample were compared with those of authentic bacillibactin or petrobactin standards. The intracellular bacillibactin and petrobactin quantities were calculated in µg/mg of cell protein. The extracellular bacillibactin and petrobactin concentrations were calculated in µg/ml of culture supernatant. Quantifications were performed three times for each extract, and means and standard deviations were calculated form the total data set.
LC-MS analysis. LC was performed on a Shimadzu LC-20AD high-performance liquid chromatography (HPLC) system consisting of a UV/visible detector (SDP-20AV) and an autosampler (SIL-20AC). The HPLC was coupled to a Shimadzu LCMS-2010EV mass spectrometer with an electrospray ionization interface. LC was carried out on an analytical column (Waters XBridge C18; particle size, 3.5 µm; column dimensions, 2.1 by 150 mm) using a linear stepwise gradient from 10% to 100% aqueous acetonitrile in 0.1% (vol/vol) formic acid at a flow rate of 0.1 ml/min over 30 min for intra- and extracellular bacillibactin analysis. Solvent systems with a gradient from 5% to 50% aqueous acetonitrile in 0.1% (vol/vol) formic acid over 30 min were used for intra- and extracellular petrobactin analysis under the LC conditions described above. The electrospray ionization source was set at the positive mode. Selected ion monitoring (SIM) was conducted to monitor ions at m/z 883.2, 719.3, and 155.2, which corresponded to the protonated molecular ions of bacillibactin, petrobactin, and internal standards 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid, respectively. The MS operating conditions were optimized as follows: drying gas flow rate, 0.1 ml/min; curved desolvation line temperature, 250°C; heat block temperature, 200°C; detector voltage, 1.5 kV.
Methylglyoxal sensitivity assays. (i) MIC.
Sterne cells were grown in LB medium to an OD600 of 0.5 and back-diluted to an OD600 of 0.01 into fresh medium containing 0, 100, 200, 300, or 500 µM methylglyoxal (Sigma M0252). Cells were allowed to grow overnight and were scored for growth versus no growth.
(ii) Disk diffusion assay. Two separate B. anthracis Sterne cultures were grown in LB medium to an OD600 of 0.7 to 0.9, and each culture was diluted 1:2 into fresh medium twice. Paraquat was added to a final concentration of 800 µM to one of each set of biological samples. Cells were grown at 37°C with shaking at 300 rpm for 45 min. Cultures were diluted 1:10 into phosphate-buffered saline, and 200 µl was spread as a lawn onto brain heart infusion agar plates. Plates were allowed to dry for 30 min, and two disks infused with 10 µl of 20% methylglyoxal were placed on each plate. H2O was used as a negative control. Plates were incubated overnight at 37°C, and zones were measured independently by two investigators in the morning. Averages were calculated for 20 disks total for each treatment (LB medium alone versus LB medium plus paraquat). A two-tailed Student t test with unequal variance was performed on Excel 2004 for Mac and on Prism 4 for Mac.
Microarray data accession number. All microarray data described in this study are freely available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-1043. The custom B. anthracis microarrays can be purchased from Affymetrix with permission from the developers (for research purposes); further information can be obtained by contacting the authors.
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RESULTS AND DISCUSSION |
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sodA1) in order to ascertain the character of the transcriptional program in response to a potentially toxic physiological perturbation of intracellular redox balance and to elucidate the impact of a lack of sodA1 on transcriptional regulation during aerobic growth. Figure 1A shows typical growth curves of B. anthracis Sterne and the sodA1 mutant in rich LB broth, indicating that these two strains have identical growth kinetics when grown in rich medium. The transcriptional responses of other bacteria to various redox cycling reagents have been explored in a variety of ways, and approaches have differed widely in the amount of time for which a given organism is exposed to a given stress (e.g., 20 min, 30 min, 2 h, 15 min, or 10 to 20 min of exposure to H2O2, and 45 min of exposure to paraquat) (14, 15, 50, 64, 66, 69). We chose to collect RNA at 20 to 25 min after exposure to two different concentrations of paraquat (100 µM and 800 µM) for several reasons. First, we wanted to perform at least three biological replicates at two different concentrations; hence, considering the number of GeneChips needed for appropriate controls, RNA was collected at one time point. Second, the two paraquat concentrations represent both a low-end concentration that is entirely nontoxic to wild type B. anthracis and only mildly attenuating to the sodA1 mutant and an upper-end concentration that is toxic only to the sodA1 mutant, but not immediately so, such that quality RNA could still be isolated from this mutant and dose-dependent levels of transcriptional change might be seen (62). Last, B. anthracis has a doubling time of approximately 50 ± 10 min in LB medium (data not shown), and because two different concentrations of paraquat were to be compared, an incubation time equivalent to one-half the doubling time was chosen in order to reveal an overall global set of transcriptional differences after two levels of an intracellular physiological imbalance were established. Our goal here was not to define a canonical "regulon" but rather to determine a global transcriptional snapshot resulting from a perturbation in intracellular physiology for two strains that we presumed would respond to the stress uniquely. We performed three independent microarray experiments for each condition utilizing three separate wild-type Sterne and sodA1 spore stock starter cultures in the absence (control) or presence (experimental) of two concentrations of paraquat (100 µM or 800 µM) (Fig. 1B) (62). Although it has been shown that the transcriptional responses to various redox cycling reagents can be rapid and differ over time (38, 80), the number of transcriptional differences we observe here show that after 20 to 25 min of exposure to paraquat, B. anthracis is undergoing an obvious transcriptional adjustment; however, we acknowledge that those genes whose transcription and RNA degradation both occur before 20 to 25 min of exposure will be missed (Fig. 1A).
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2-fold). We are primarily interested in genes whose expression is induced by superoxide stress; however, all genes at least twofold differentially expressed, both up- and down-regulated, are listed in the tables in the supplemental material, arranged by TIGR gene number. Significantly overrepresented functional families were identified using the EASE algorithm as stated in Materials and Methods (39).
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sodA1 mutant relative to the parental Sterne strain during exponential growth in rich medium in the absence of induced oxidative stress.
Because sodA1 was found to be necessary for resistance to superoxide stress induced by the compounds paraquat and menadione (62), we hypothesize that this gene is important for the general maintenance of the redox status of the cell during normal aerobic growth in vitro, when superoxide is expected to be generated spontaneously during electron transport. Therefore, we compared genes differentially expressed by the sodA1 mutant relative to the wild-type parental strain during growth in rich medium without induced oxidative stress. SAM reveals that 25 genes are significantly overexpressed by the sodA1 mutant, whereas 113 are significantly underexpressed (Table 3, last column; see also the tables in the supplemental material). The low ratio of overexpressed versus underexpressed genes (0.22 in this comparison, versus 1.3 for Sterne and 2.4 for the sodA1 mutant in response to 800 µM paraquat) is notable because underexpression of genes cannot be explained merely by slower growth, since this mutant grows equivalently to Sterne in LB medium (Fig. 1A). Many of the genes underexpressed by the sodA1 mutant encode proteins with unknown biological functions, a common challenge encountered in many microarray experiments. Functional-family analysis (Table 2, last column) shows that even without induced superoxide stress, the sodA1 mutant expresses several transport and binding genes and metabolic genes at higher levels than Sterne, suggesting that this mutant is undergoing endogenous superoxide stress or an undefined oxidative stress simply as a result of normal metabolism, without the addition of a redox cycling compound, and that this stress does not result in a lower growth rate.
One prominent group of genes overexpressed by the sodA1 mutant during logarithmic growth consists of GBAA1778, -1779, and -1780 (up-regulated 49-, 23-, and 31-fold, respectively). An operon prediction algorithm developed by Bergman et al. (5) predicts that the first two genes are most certainly transcribed as one unit, whereas inclusion of the third gene is less certain. End point RT-PCR using primers designed to amplify overlapping gene regions showed that these three genes are, in fact, transcribed as one unit (data not shown). It can reasonably be assumed that the first two genes, a putative cytochrome c-type biogenesis protein (ccdA-1) and a putative thioredoxin family protein, are related to the general cellular redox status of the cell. Homologs of these two genes are located together on the genomes of diverse microorganisms such as Streptococcus pyogenes, Clostridium tetani, Dehalococcoides ethenogenes, and Thermoanaerobacter tengcongensis (genome region comparison performed at http://cmr.tigr.org). However, the third protein, a putative prolipoprotein diacylglyceryl transferase, is located just downstream of the other two only in the pathogenic Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) as far as the current TIGR database of sequenced genomes can reveal. The polycistronic nature of these proteins in the pathogenic Bacillus cereus group is unknown and may merit further investigation. This example highlights the way in which combining various bioinformatic approaches can reveal potentially interesting phenomena that may not be observed otherwise.
The remaining genes expressed at higher levels by the sodA1 mutant during normal growth encode a variety of proteins, including menaquinone (GBAA5111), uridylate kinase (pyrH; GBAA1797), a putative membrane protein (GBAA1904), acid phosphatase (GBAA4746), DNA topoisomerase III (topB-2; GBAA1905), various transporters, and others (Tables 2 and 3, last columns); most showed moderate increases in expression, ranging from two- to fourfold relative to wild-type levels. The only other gene whose expression was notably increased to very high levels in the sodA1 mutant during normal growth encodes an M23/M37 family peptidase. It is reasonable to predict that the sodA1 mutant may harbor higher levels of protein damage from oxidative stress during aerobic growth than the parental strain. This topic is currently under investigation.
The relatively high expression levels of these genes in the sodA1 mutant compared to the parental strain without paraquat stress highlight the complex, multifactorial ways in which even minor changes in intracellular redox conditions (i.e., as a result of the lack of a major antioxidant enzyme) can affect gene regulation. The data below, on the other hand, showing differences in gene expression after treatment with different concentrations of paraquat, most likely reflect changes in gene regulation due to multiple downstream effects caused by an initial increase in intracellular superoxide levels imposed by paraquat. For example, because SODs themselves generate H2O2 during their enzymatic dismutation of superoxide, and since the sodA1 mutant lacks the most effective form of SOD, wild-type cells may potentially be responding to higher levels of H2O2. Superoxide itself can cause various effects (including protein damage, metal ion stability, accumulation of H2O2 due to the action of SOD proteins, impaired enzymatic functions of redox sensitive enzymes, etc.), and the various transcriptional differences seen in treated versus untreated cells most likely reflect various levels of shifts in homeostasis. Regardless of the exact mechanisms, the global changes reflect the ways in which B. anthracis has evolved to handle both major and minor shifts in intracellular redox conditions. The annotated genomes of B. anthracis strains have no homolog to the well-defined E. coli soxR soxS superoxide-sensitive regulatory system (65), and the various regulatory networks of B. anthracis have been only partially characterized (31).
Functional-family analysis and SAM comparing gene families and individual genes expressed differentially after exposure to paraquat. (i) Functional families.
Functional-family analysis is a useful way to view the overall, end point character of an induced transcriptional response. Table 2 lists the families of genes that are significantly overrepresented within the subset of B. anthracis genes that change significantly after treatment with paraquat. The most evident trend is the large number of transport and binding protein genes and siderophore biosynthesis genes more highly expressed upon paraquat exposure, suggesting that oxidative stress is tightly correlated with metal ion homeostasis. It should be noted that very few transport proteins have been characterized in B. anthracis (33). However, it is clear that (i) a higher dose of paraquat induces the transcription of a greater number of putative transport proteins in Sterne, and (ii) at both doses, the sodA1 mutant expresses at least threefold more genes in this category than Sterne, once again suggesting that sodA1 maintains intracellular redox conditions that affect gene transcription.
The transcriptional response of the sodA1 mutant to paraquat was exaggerated compared with that of the parental Sterne strain (Tables 2 and 3, compare Sterne to sodA1 columns). The Sterne strain responded to 100 µM paraquat by inducing the expression of 19 genes, with no genes underexpressed, while the sodA1 mutant increased the transcription of 119 genes twofold or more and decreased the transcription of 17 genes. The difference in the increase in transcription between these two strains (sixfold higher for the sodA1 mutant) highlights the importance of sodA1 in maintaining levels of intracellular redox homeostasis that may result in the transcription of many genes. Addition of the higher concentration of paraquat (800 µM) resulted in a larger number of genes differentially expressed in Sterne (59 up-regulated; 45 down-regulated), while the sodA1 mutant again responded more strongly (154 up-regulated; 64 down-regulated). Note that the increased transcription in the sodA1 mutant in response to the low dose of paraquat was still substantially higher than that of Sterne in response to an eightfold-higher dose, once more stressing the importance of sodA1 to intracellular redox maintenance. Also note that several genes were detected as more highly expressed in the sodA1 mutant at the lower dose of paraquat and not at the higher dose (Table 3, sodA1 columns). Unlike those of the Sterne strain, the growth kinetics of the sodA1 mutant are affected at this concentration of paraquat: upon addition of 800 µM paraquat to logarithmically growing cells, the rise in OD600 levels off and then begins to decrease after about 4 h of exposure, showing that this concentration of reagent eventually proves lethal to this mutant (62). Hence, it is not surprising that some unexpected trends may be observed at this dose with the mutant strain. The majority of genes induced in the sodA1 mutant, however, are detected after treatment with both doses of paraquat at levels well above twofold relative to the control, and many of these genes have very clear connections to redox functions [e.g., NAD(P)H dehydrogenase, flavodoxins, and nitroreductase family protein (Table 3)].
Curiously, none of the B. anthracis sod genes showed increased expression after 20 to 25 min of paraquat treatment. It is possible that a transient increase in expression may have occurred before RNA harvesting in these experiments. However, expression of the sod genes during logarithmic growth and entry into sporulation phase showed that three of the four sod genes are, for the most part, expressed constitutively (62). Ongoing studies also show that, in contrast to the pattern for E. coli, neither sodA1 nor sodA2 is expressed at higher levels in B. anthracis growing in iron-depleted medium than in rich medium (22, 23; also data not shown), and it is possible that B. anthracis has evolved less complex sod regulation than other bacteria. However, a study of B. anthracis gene expression during infection of macrophages identifies sodA2 as showing increased expression during intracellular growth (N. H. Bergman et al., submitted for publication), an intriguing finding given that this isoform of the enzyme is not a major player in protection from oxidative stress and is not needed for survival in the susceptible mouse model (62).
(ii) Glyoxalase system.
Notably, the expression of three separate putative glyoxalase family genes is greatly increased in both Sterne and the sodA1 mutant upon treatment with paraquat at both doses (increases of 37- to 85-fold for GBAA1653, 3- to 28-fold for GBAA3339, and 4- to 18-fold for GBAA3878 [Table 3]). The glyoxalase cycle is highly conserved in eukaryotes and prokaryotes, is important in certain types of cellular detoxification (16), and has been linked to potassium efflux in E. coli (51). The nucleophile methylglyoxal is a toxic but naturally occurring metabolite that can accumulate in cells as a result of various metabolic processes (9, 27), and the glyoxalase system has evolved as a protective mechanism. The major protective glyoxalase system in E. coli is glutathione dependent (51), but it is believed that B. anthracis does not use glutathione in cellular redox reactions (25), so the glyoxalase system(s) in B. anthracis is presumably non-glutathione dependent. Note that the annotated B. anthracis Ames Ancestor genome lists 18 unique "glyoxalase family proteins" (6 of which are spelled "glyoxylase"), further highlighting the high degree of potential redundancy encoded on the genome, a phenomenon that has been observed for this bacterium before (37).
Protein sequence comparisons reveal that the three B. anthracis glyoxalase paralogs identified in this study are homologous mainly to other gram-positive microorganisms such as Bacillus, Staphylococcus, and Oceanobacillus species. The disparate locations of the three genes on the chromosome and the concomitant change in expression in response to paraquat suggest a conserved regulation of the genes that is sensitive to superoxide or to downstream effects caused by rises in superoxide levels. We therefore investigated the toxicity of the compound methylglyoxal to B. anthracis Sterne cells before and after paraquat treatment. We found that priming vegetatively growing Sterne cells with 800 µM paraquat for 45 min in LB medium causes the cells to be slightly more resistant to methylglyoxal in a disk diffusion assay than cells grown without paraquat. A preliminary experiment demonstrating this trend led us to repeat the experiment with two independent starter strains and a larger number of replicates. The average zone of inhibition of untreated Sterne cells was 25.5 mm (standard deviation, ±0.9), whereas cells that had been grown in the presence of 800 µM paraquat had an average zone diameter of 22.1 mm (standard deviation, ±0.7) (n = 20 for each condition, paraquat treatment or no treatment; P < 0.0001 by an unpaired, two-tailed Student t test; zones were measured independently by two investigators). The MIC of methylglyoxal for logarithmically growing cells that are back-diluted to an OD600 of 0.01 is between 200 µM (strong growth) and 300 µM (no growth). The ability of the paraquat-primed cells to be slightly more resistant to methylglyoxal suggests that at least part of the glyoxalase system in B. anthracis responds to intracellular redox stress. A putative glyoxalase has been identified as potentially contributing to the physiology of the major pathogen Mycobacterium tuberculosis (40). Also, studies of B. anthracis gene expression during macrophage infection indicate an increase in the expression of two putative glyoxalases (Bergman et al., submitted), different from those identified in this study, further highlighting the potentially multifaceted roles for glyoxalases in bacterial metabolism and virulence.
(iii) Siderophore biosynthetic genes and putative transporters. Siderophores are small metabolites synthesized by bacteria to scavenge iron from the extracellular milieu during iron limitation, and iron metabolism has been correlated with the virulence of various pathogens (10, 77). The B. anthracis siderophore biosynthetic genes up-regulated in response to paraquat, with one exception, were in the operon containing homologs to the B. subtilis bacillibactin biosynthetic genes (the dhb operon; the B. anthracis operon is referred to below as the bac operon) (53). The five B. anthracis genes GBAA2368 to -2373 share 60 to 70% protein identity with the B. subtilis homologs. It has been shown that B. anthracis produces the bacillibactin metabolite at low levels (48), but the major siderophore that B. anthracis uses for survival under iron-limiting conditions is a second molecule called petrobactin, which is synthesized by genes included in the asb operon (13, 49, 79). Up-regulation of bacillibactin biosynthetic genes and of other genes under the transcriptional control of the iron-responsive regulator fur in response to oxidative stress has been seen in B. subtilis (58). The regulation of the homologous genes in B. subtilis by the iron-responsive Fur element has been studied extensively (2, 53, 58, 61). However, the four putative fur family and iron-dependent regulators in B. anthracis have not been characterized, so direct correlation between fur regulation and oxidative stress cannot be verified here. The canonical fur binding sequences upstream of the bac operon, however, are identical in B. subtilis and B. anthracis, whereas the binding sequence upstream of the asb (petrobactin biosynthetic) operon is not entirely conserved (data not shown). This finding and the fact that, in contrast to the results for the bac operon, transcription of only one of the asb genes was shown to be slightly increased after paraquat treatment strongly suggest that these two biosynthetic operons are not subject to identical transcriptional control, at least under superoxide stress.
The bacillibactin biosynthetic gene cluster in B. subtilis is composed of five adjacent genes termed dhbACEBF (2, 12, 53, 67). The homologs in B. anthracis maintain this gene order (GBAA2368 to -2372), but in B. anthracis, three downstream genes (GBAA2373, -2374, and -2375) that are not present in the B. subtilis dhb region are also overexpressed after paraquat treatment at levels roughly equivalent to those observed for GBAA2368 to -2372 (Table 3, siderophore biosynthesis). Homologs of these three genes are often found near nonribosomal peptide synthesis operons in other bacteria (73), and the operon prediction algorithm described by Bergman et al. (5) predicts that cotranscription of these three genes in B. anthracis is highly probable. End point RT-PCR probing for overlapping transcription products confirms that these genes are included in the B. anthracis bac transcriptional unit (data not shown), further highlighting differences between B. subtilis and B. anthracis. The three genes encode an MbtH-like protein, an EmrB/QacA family drug-resistance transporter, and a putative 4'-phosphopantetheinyl transferase. The MbtH-like protein has approximately 56% identity with the M. tuberculosis homolog by ClustalW alignment, and homologs of this protein are found near nonribosomal peptide synthetases (73) in other bacteria. Although this small protein (70 amino acids) is part of the operon for the biosynthesis of the very important mycobactin siderophore of M. tuberculosis, its function has not been determined (17). The connection with bacillibactin implied by the cocistronic structure suggests that these genes have an alternative function that has not yet been ascertained, or that they, too, are involved in the biosynthesis of bacillibactin in a way that is not conserved or not transcriptionally coupled in B. subtilis.
The last section of Table 3 lists the many putative transport and binding proteins that show increased expression after paraquat stress, primarily in the sodA1 mutant. Of the putative transporters that showed increased expression in response to paraquat only in the sodA1 mutant, many are annotated as putative iron compound transporters, and some of these show very large increases (up to 69-fold) in expression. Because these transporters are currently uncharacterized, it is difficult to know whether they are specific for iron and whether they are responsible for metal influx or efflux. Regardless, the large number of genes with a putative iron transport role suggests a very large shift in iron homeostasis in the sodA1 mutant in response to paraquat (see "Growth of multiple B. anthracis sod mutants in various metal-limiting media" below). Three general permease/major facilitator family transporter genes showed substantially increased expression in both Sterne and the sodA1 mutant after both paraquat treatments (39- to 147-fold for GBAA1652, 10- to 75-fold for GBAA2346, and 6- to 13-fold for GBAA5260 [Table 3]), suggesting that the expression of these genes is highly sensitive to intracellular superoxide levels or is somehow caused by the damage resulting from a rise in superoxide levels.
To confirm the increase in the expression of select genes that we observed with the microarrays, we performed SYBR green quantitative RT-PCR on a small subset of genes with fresh RNA samples (Table 4). As in the microarray experiments, a region within the bac operon was seen to be induced substantially after treatment with paraquat, whereas the expression of a gene from the asb operon was increased only slightly in Sterne and more in the sodA1 mutant. Additionally, the glyoxalase family gene GBAA3339 was seen to undergo a major increase in expression in response to paraquat. Lastly, the ccdA-1 operon transcript was assayed from strains growing in LB alone, and the sodA1 mutant transcribed these genes at substantially higher levels than the parental Sterne strain during normal exponential growth. It should be noted that n-fold changes are not expected to be identical for the SYBR green assays and the microarrays, since normalization in the microarrays is extremely stringent, detecting 18 separate probes for each open reading frame, making for a much more robust system than the use of a single primer pair per gene as in the RT-PCR.
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bac mutants grow as well as the wild type during iron limitation (13; also data not shown). Table 3 shows that the induction of the bac genes in response to paraquat was substantial in Sterne (ranging from 5- to 12-fold) and even more prominent in the sodA1 mutant (ranging from 42- to 163-fold). Of the asb biosynthetic genes, only one was more than twofold induced in response to 800 µM paraquat in the sodA1 mutant.
Koppisch et al. (48) report that petrobactin is the main siderophore produced in iron-limiting medium, with relatively low levels of bacillibactin produced. However, they observed that cultures grown in ambient air seemed to produce higher levels of bacillibactin. Quantities of intra- and extracellular bacillibactin and petrobactin from B. anthracis were monitored using LC-MS by isolating supernatants and lysates of exponentially growing cells of the Sterne, sodA1, and bac strains in iron-rich medium (LB) and IDM, with or without the addition of paraquat (Table 5). We note that siderophores are intricate metabolites, and the biosynthesis of these molecules is highly complex, involving the cooperation of a number of specific enzymes and cofactors to assemble them; hence, considering the complexity of these systems, expression of the biosynthetic proteins, construction of macromolecular complexes, and the appearance of the metabolite could be temporally distinct rather than coincident (for an overview of the siderophore "assembly line," see reference 17). Therefore, we isolated cell supernatants and lysates 1 h after paraquat treatment to allow time for protein translation and metabolite synthesis to occur. The LC-MS analysis revealed that neither bacillibactin nor petrobactin accumulated in supernatants or lysates of cells grown in iron-rich LB medium, with or without paraquat (data not shown). Intracellular bacillibactin accumulated in Sterne and the sodA1 mutant grown in IDM but was not detected in sodA1 lysates after paraquat treatment and was just barely detected in lysates of Sterne grown in the presence of paraquat. However, bacillibactin was detected in supernatants of Sterne and the sodA1 mutant grown in IDM, both with and without paraquat, but counter to what the transcriptional data suggest, the levels of bacillibactin were substantially decreased in paraquat-treated cells. A bac mutant served as a negative control, and no bacillibactin was detected in this strain under any conditions. These data suggest that the expression of the bac genes in response to oxidative stress may be subject to additional layers of regulation (posttranscriptional) or that the added intracellular oxidative stress caused by paraquat may prevent various redox-sensitive enzymatic processes from occurring during the complex, multistep production of bacillibactin (17, 53). The bac mutant shows sensitivities to paraquat and H2O2 identical to those of the parental Sterne 34F2 strain (data not shown), and thus bacillibactin does not appear to be playing an overt role in protection from oxidative stress. However, the conservation of a functional bacillibactin biosynthetic pathway may indicate an alternative role for bacillibactin in B. anthracis physiology that has not yet been observed, or it may indicate that bacillibactin has a mildly redundant but minor role in iron acquisition compared to petrobactin. Recently, it was shown that certain proteins of host cells are able to scavenge and bind bacillibactin but are less efficient at sequestering petrobactin, highlighting the role of the petrobactin biosynthetic cluster in the evolution of virulence (1).
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sodA1, and bac strains as well. Substantial levels of petrobactin were isolated from IDM culture supernatants of all strains, with or without paraquat, and as with bacillibactin, levels of petrobactin were decreased after paraquat treatment, again suggesting that enzyme function during siderophore production may be compromised by intracellular redox stress. But petrobactin was never detected in cell lysates (Table 5; Fig. 2), suggesting that petrobactin synthesis and export may be tightly coupled. The study of siderophore transport has focused mainly on the process of metal import (11, 26, 55), while the process of siderophore export has been studied less extensively, and primarily for the gram-negative organism E. coli (7, 32). A spatial study of siderophore biosynthesis and export would be of great interest. It should be noted that bacillibactin and petrobactin were also quantified by HPLC from cells grown to stationary phase for 24 h (data not shown) and that these data showed the same trend of decreased siderophore concentrations after paraquat treatment.
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sod mutants in various metal-limiting media.
Iron homeostasis and oxidative stress are often linked biologically (60, 71, 72, 78), and the increased expression of the bacillibactin biosynthetic operon and numerous putative, uncharacterized transporters after paraquat stress that we observed was suggestive of an iron starvation response, especially for the sodA1 mutant. Although high intracellular iron levels in the cytoplasm can be toxic, especially in the presence of reactive oxygen, where they can lead to the formation of very damaging hydroxyl radicals (41), evidence from E. coli suggests that a lack of SOD can, counter to intuition, lead to an iron starvation phenotype, even in the presence of ample cytoplasmic iron (52). Maringanti and Imlay propose that in the presence of superoxide, E. coli sod mutants acquire damage to various iron-containing proteins, leading to the release of free iron that is "not usable," causing the bacteria to import new, "usable" iron for the repair of damaged enzymes, all the while accumulating potentially toxic levels of "unusable" intracellular free iron (52). With this precedent in mind, we tested the growth of several B. anthracis sod mutants in a variety of iron/cation-limiting media to determine if these mutants have a higher need for iron/cation transport. This question is of particular interest due to the fact that the host environment is believed to be both iron limiting and potentially oxidatively challenging. We tested bacterial growth in two different types of iron-limiting media: chelated and depleted. Chelated medium has the cation chelator (in this case, 2,2'-dipyridyl) present at all times, in effect binding metal and preventing its uptake in a concentration-dependent manner, whereas depleted medium has had the majority, but not the entirety, of metal salts preremoved with a resin (Bio-Rad Chelex 100) and has then been supplemented with several metal salts (e.g., magnesium and/or manganese). Consequently, chelated medium can effectively shut or slow down transport by holding ions unavailable, whereas depleted medium (IDM or IMnDM) allows ample transport, but with smaller amounts of various ions present for utilization.
Figure 3 depicts growth curves of multiple B. anthracis strains in LB medium containing either no chelating agent or the metal-chelating compound 2,2'-dipyridyl at 300 or 400 µM. The asb mutant strain, which does not produce the siderophore petrobactin (79) and has been shown to have a growth defect in iron-poor medium (13, 49), was included as an indicator of iron depletion. Also included in these experiments were the sodA2 strain, lacking the second active B. anthracis SOD, and the sodA1 sodA2 strain, lacking both active SODs.
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asb strain is most affected, confirming the importance of the siderophore petrobactin for B. anthracis under extremely iron poor conditions. The parental strain, Sterne, and the sodA2 mutant grow identically and to a lower final OD600 in the presence of 2,2'-dipyridyl. As with E. coli (52), the B. anthracis sodA1 mutant and the sodA1 sodA2 double mutant show a consistent growth defect in chelated medium, supporting the idea that only sodA1 is needed for optimal physiological superoxide homeostasis during iron limitation and that no redundancy exists between sodA1 and sodA2 (62). Additionally, the MICs of 2,2'-dipyridyl that prevent the outgrowth of spores of the sodA1 and sodA1 sodA2 mutants are between 200 and 300 µM, whereas the MICs for spores of Sterne and the sodA2 mutant are above 500 µM, with outgrowth sometimes occurring for these strains above 500 µM (data not shown).
Because 2,2'-dipyridyl binds multiple cations in a complex manner, we chose to further explore the growth of the sod mutants in two different depleted media, where low concentrations of metal ions are available for transport and the presence of specific metal ions can be manipulated. IMnDM was prepared as described in Materials and Methods and previously (13). When ferrous iron (Fe) and manganese (Mn) were added back into IMnDM, all strains grew equivalently (Fig. 4A). Surprisingly, in IMnDM with no Fe or Mn supplementation, only the sodA1 sodA2 double mutant showed a severe growth defect, while even the asb mutant showed only mild attenuation. The ability of the asb mutant to eventually "catch up" to Sterne in IMnDM, compared to its severe growth defect in chelated medium, suggested that iron is still somewhat present and available even after resin depletion. Mn supplementation alone caused total recovery for the sodA1 sodA2 double mutant but not for the asb mutant (Fig. 4C), while Fe supplementation alone allowed recovery only for the asb mutant (Fig. 4D), confirming that these two phenotypes are Mn and Fe specific, respectively. Growth in IMnDM of complementation strains of the sodA1 sodA2 double mutant, expressing the wild-type sodA1 or sodA2 gene on a cytoplasmic plasmid (Fig. 4E and F), showed that (i) the presence of sodA1 is necessary for the most robust growth during Mn limitation; (ii) the presence of sodA2 allows for a slightly lower level of recovery; and (iii) the absence of both enzymes causes a severe growth defect in low-Mn medium. The sodA1 sodA2 double mutant is identical to the sodA1 single mutant in terms of sensitivity to paraquat and diamide (data not shown), and these results represent the first instance where the two mutants have exhibited differing behavior and suggest a mild redundancy for sodA1 and sodA2. The sodA2 gene is actively transcribed and encodes an active SOD enzyme (62), so a level of redundancy would be expected between these two enzymes. These data, however, still support the preeminence of sodA1 as the more effective and/or more utilized of the two enzymes. Since iron is only slightly limiting in IMnDM, the possibility exists that one or both of the SODs might be cambialistic (able to utilize Mn or Fe as a catalytic ion). Regardless, as with E. coli, the B. anthracis sodA1 and sodA1 sodA2 mutants behaved as if they were iron starved, and the presence of various intracellular iron pools under oxidative stress is currently being investigated.
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sodA1 mutant strains to different levels of intracellular superoxide stress generated endogenously by the compound paraquat. This global approach identified various groups of genes that showed increased transcription after different levels of oxidative stress in two strains that are differentially sensitive to paraquat. First, a small set of genes showed increased transcription in the sodA1 mutant relative to the parental strain during normal growth in rich broth, independently of induced chemical stress. Because most of these genes were not induced in the parental strain after paraquat stress, their regulation may be controlled by an as yet unidentified, paraquat-independent effect that a lack of sodA1 may facilitate. Next, a group of genes showed increased expression in Sterne in response to a low concentration of paraquat (100 µM); all these genes showed even higher induction in Sterne after exposure to 800 µM paraquat, and all of them were induced under both conditions in the sodA1 mutant. We propose that these genes may be under the control of a regulatory system that is quite sensitive to the presence of superoxide or to the damage caused by this reactive metabolite. Still other genes were induced only at the higher concentration of paraquat in Sterne and at both concentrations in the sodA1 mutant, likely representing a level of regulation less sensitive to superoxide stress than that for the previous group. Lastly, we observed an increase in the transcription of many genes only in the sodA1 mutant at both concentrations of paraquat; these genes may be under the control of regulatory systems that respond only to very high levels of superoxide or oxidative damage that can be metabolically controlled when sodA1 is present. The data presented here highlight the intrinsic and often contradictory connection between oxidative stress and metal acquisition in bacteria and indicate that B. anthracis has evolved multiple mechanisms to cope with these problematic conditions, which may be encountered within the host. We hope that the trends elucidated here will aid in the further investigation of the metabolic and regulatory processes used by the important pathogen B. anthracis.
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ACKNOWLEDGMENTS |
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Funding for this work was provided by the University of Michigan Cellular Biotechnology Training Program, the University of Michigan F. G. Novy Fellowship, HHS contract N266200400059C/N01-A1-40059, NIH grant AI08649, and the Great Lakes Center of Excellence for Biodefense and Emerging Infections.
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FOOTNOTES |
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Published ahead of print on 23 March 2007.
Supplemental material for this article may be found at http://jb.asm.org/.
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