Toxicogenomic response of Mycobacterium bovis BCG to

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Toxicogenomic response of Mycobacterium bovis BCGto peracetic acid and a comparative analysis of the M. bovisBCG response to three oxidative disinfectants

Chantal W. Nde & Freshteh Toghrol & Hyeung-Jin Jang &

William E. Bentley

Received: 1 June 2010 /Revised: 8 September 2010 /Accepted: 1 October 2010# Springer-Verlag (outside the USA) 2010

Abstract Tuberculosis is a leading cause of death world-wide and infects thousands of Americans annually. Myco-bacterium bovis causes tuberculosis in humans and severalanimal species. Peracetic acid is an approved tuberculocidein hospital and domestic environments. This study presentsfor the first time the transcriptomic changes in M. bovisBCG after treatment with 0.1 mM peracetic acid for 10 and20 min. This study also presents for the first time acomparison among the transcriptomic responses of M. bovisBCG to three oxidative disinfectants: peracetic acid,sodium hypochlorite, and hydrogen peroxide after 10 minof treatment. Results indicate that arginine biosynthesis,virulence, and oxidative stress response genes wereupregulated after both peracetic acid treatment times. ThreeDNA repair genes were downregulated after 10 and 20 minand cell wall component genes were upregulated after20 min. The devR–devS signal transduction system wasupregulated after 10 min, suggesting a role in the protection

against peracetic acid treatment. Results also suggest thatperacetic acid and sodium hypochlorite both induce theexpression of the ctpF gene which is upregulated inhypoxic environments. Further, this study reveals that inM. bovis BCG, hydrogen peroxide and peracetic acid bothinduce the expression of katG involved in oxidative stressresponse and the mbtD and mbtI genes involved in ironregulation/virulence.

Keywords Microarrays .Mycobacterium bovis BCG .

Peracetic acid . Sodium hypochlorite . Hydrogen peroxide .

Transcriptomics

Introduction

Despite extensive research that has been carried out and theavailability of effective chemotherapy, tuberculosis (TB)still remains a leading cause of death worldwide (Sassetti etal. 2003). Data from the Centers for Disease Control andPrevention (CDC) indicate that by the end of 2007, twobillion people worldwide were infected by Mycobacteriumtuberculosis, which is the most common cause of TB in theUSA. CDC reports also indicate that although the numberof TB cases is on the decrease in the USA, thousands ofAmericans are still infected and hundreds die annually fromthe disease.

M. tuberculosis and Mycobacterium bovis are morethan 99% genetically similar (Garnier et al. 2003). M. bovisis part of the M. tuberculosis complex and is implicated intuberculosis infections in humans and several animalspecies (Garnier et al. 2003; Golby et al. 2007). Thetuberculosis vaccine strain M. bovis Bacillus Calmette-Guerin (M. bovis BCG) was derived from M. bovis (Garnieret al. 2003; Keller et al. 2008).

Electronic supplementary material The online version of this article(doi:10.1007/s00253-010-2931-6) contains supplementary material,which is available to authorized users.

C. W. Nde :W. E. BentleyCenter for Biosystems Research,University of Maryland Biotechnology Institute,College Park, MD 20742, USA

F. Toghrol (*)Microarray Research Laboratory, Biological and EconomicAnalysis Division, Office of Pesticide Programs,U.S. Environmental Protection Agency,Fort Meade, MD 20755, USAe-mail: [email protected]

H.-J. JangCollege of Oriental Medicine, Kyung Hee University,Seoul, South Korea

Appl Microbiol BiotechnolDOI 10.1007/s00253-010-2931-6

http://dx.doi.org/10.1007/s00253-010-2931-6

Oxidative disinfectants including peracetic acid, sodiumhypochlorite, and hydrogen peroxide are approved by theEnvironmental Protection Agency as active ingredients indisinfectants used for the eradication of pathogens includingM. tuberculosis in the hospital, domestic, and agriculturalenvironments. Studies have reported that peracetic acidtreatment leads to protein and enzyme denaturation andincreased cell wall permeability of bacteria through thedisruption of sulfhydryl (–SH) and disulfide bonds (S–S)(Kitis 2004; Block 2001). We have also previously shownusing microarray technology that in Staphylococcus aureus,peracetic acid alters the expression of membrane transportgenes and induces the transcription of DNA repair andreplication genes (Chang et al. 2006b). To our knowledge,our recent studies of the toxicogenomic response of M. bovisBCG to sodium hypochlorite and hydrogen peroxide are theonly studies that report the global transcriptomic response ofany mycobacterial species to oxidative disinfectants (Jang etal. 2009a, b). Peracetic acid is approved for disinfectionagainst mycobacteria, yet the mechanism of action ofperacetic acid in any mycobacterial species from a globalgenomic perspective has not been investigated.

Several previous reports have compared microarraystudies, either for analyzing the responses of one organismto different treatments or the responses of differentorganisms to the same treatment. However, the results ofthese studies are often indecisive (Small et al. 2007). Acomparative analyses of the global transcriptomic effects ofdifferent antimicrobials in the same pathogenic organismwill provide information on major differences and similaritiesbetween the metabolic pathways affected in that organismfollowing treatment with these disinfectants. This informationwill help in the identification of commonly regulated genes inresponse to these antimicrobials which will facilitate theunderstanding of their modes of action. The current study andour previous studies (Jang et al. 2009a, b) have detailed thetoxicogenomic response of M. bovis BCG to peracetic acid,sodium hypochlorite, and hydrogen peroxide. Using the datafrom these reports, we carried out a comparative analysis ofthe transcriptomic responses observed after 10 min oftreatment of M. bovis BCG with the three oxidativedisinfectants. We focused on data generated after 10 min oftreatment in this analysis as it was a common treatment timeamong the three studies. A significant advantage of thecomparative analysis carried out in the second section of thisstudy is that the transcriptome data and real-time polymerasechain reaction (PCR) validation of microarray results wasobtained from experiments carried out under similarexperimental conditions in our laboratory.

In the first section of this study, we performed ananalysis of the toxicogenomic response of the modelorganism, M. bovis BCG to 0.1 mM peracetic acid usingAffymetrix M. bovis BCG custom arrays. Results from the

first section of this study identify signature genes that aredifferentially regulated in mycobacteria in response to per-acetic acid and improve the understanding of the genetic basisof resistance to this disinfectant. In addition, the informationgenerated from this study sheds more light on the mechanismof action of peracetic acid in Mycobacteria. The secondsection of this report provides the first comparative analysisof the global gene response of M. bovis BCG to oxidativeantimicrobials and improves the understanding of thesimilarities between the mechanisms of action of thesedisinfectants. In addition, this comparative analysis providesinformation that can be used for the development of moreeffective oxidative antimicrobials and antimicrobial mixtures.

Materials and methods

Preparation of bacterial culture and growth conditions

As previously described (Jang et al. 2009a, b), a stockculture of M. bovis BCG strain Pasteur 1173P2 (ATCC35748) was inoculated into 200 ml Middlebrook 7H9broth (Difco, Sparks, MO, USA) supplemented with 0.1%(v/v) Tween 80 (Sigma-Aldrich Co., St. Louis, MO, USA)and 10% (v/v) OADC (oleic acid, albumin, dextrose,catalase). Following incubation at 37°C with shaking at200 rpm, the culture reached an OD600 of 0.3–0.4 after5 days. One-milliliter volumes of this culture were maintainedin 10% (v/v) glycerol at −80°C for subsequent use.

Measurement of cellular adenosine triphosphate

Due to the characteristic slow growth of M. bovis BCG, thequantity of adenosine triphosphate (ATP) produced by cellstreated with peracetic acid as opposed to colony counts wasused to monitor the changes in the number of viable cells.A 1-ml aliquot of the prepared M. bovis culture was addedto the M7H9 medium and incubated at 37°C with shakingat 200 rpm to reach an OD600 of 0.3–0.4 after 5 days. Cellswere harvested and resuspended in 200 ml of Luria–Bertani(LB) broth containing 0.1%Tween 80 and incubated for 24 h at37°C to reach an OD600 of 0.3–0.6. The amount ofluminescence in relative light units (RLU) produced by cellswas measured using the Bac-Titer Glo™ microbial cellviability assay and the Glomax™ luminometer (PromegaCo., San Luis Obispo, CA, USA). A standard curve relatingluminescence (RLU) and the corresponding amount of ATP inpicomoles has been previously reported (Jang et al. 2009a, b).

Peracetic acid treatment and ATP measurements

To determine a value for background luminescence prior todisinfectant treatments, cells in 1 ml of the untreated culture

Appl Microbiol Biotechnol

in LB broth were harvested, washed in 1 ml 1× phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, CA, USA) andresuspended in 200 μl PBS. A 100-μl volume of theresuspended pellet of untreated cells was added to markedcontrol wells of a 96-well plate containing 100 μl of theBac-Titer Glo buffer–substrate mixture and luminescencewas measured. LB growth cultures were dispensed intodesignated 50 ml tubes, and peracetic acid was added to thecultures to reach test concentrations of 0, 0.05, 0.1, 0.2, and0.5 mM. Luminescence measurements were performed at10-min intervals during a 1-h period for the differentperacetic acid concentrations as described for the untreatedculture.

RNA extraction

M. bovis BCG cells treated with peracetic acid anduntreated cells were harvested and resuspended in PBSbuffer. The mini-bead beater-16 (BioSpec Products Inc,Bartlesville, OK, USA) was used for breaking down thecells. Beating was carried during five 1-min periods.Microcentrifuge tubes containing the cells were stored onice for 2 min after each beating period. RNA was extractedfrom untreated to peracetic acid-treated (0.1 mM) cells after10 and 20 min using the RiboPure bacteria kit (Ambion,Inc., Austin, TX, USA). Eluted RNA was quantified usingthe NanoDrop spectrophotometer (NanoDrop Technologies,Inc., Wilmington, DE, USA). RNA quality and purity werechecked using the Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA, USA).

Complementary DNA synthesis, labeling, hybridization,staining, and scanning

Complementary DNA (cDNA) synthesis, fragmentation,labeling, hybridization, washing, and staining wereperformed according to instructions for the AffymetrixGeneChip arrays (Affymetrix, Inc., Santa Clara, CA,USA) as reported in our previous publications (Chang etal. 2006a, b; Jang et al. 2008; Nde et al. 2008).

Data analysis: toxicogenomic response of M. bovis BCGto peracetic acid

Data analysis was performed using the Affymetrix GeneChipOperating Software (GCOS), version 1.0, and GeneSpringVersion 7.3 (Agilent Technologies). Parameters employed forexpression analysis using GCOS include α1=0.04, α2=0.06, τ=0.015, and target signal was scaled to 150.Statistically significant changes in gene expression wereidentified by one-way ANOVA (p value≤0.05). Foldchanges were calculated as the ratios between the signalaverages of three untreated (control) and three peracetic

acid-treated cultures. Genes with a 2-fold or moreinduction or repression were used in this analysis.

Data analysis: comparisons among the toxicogenomicresponses of M. bovis BCG to sodium hypochlorite,hydrogen peroxide, and peracetic acid

The Affymetrix GCOS, version 1.0, and GeneSpringVersion 7.3 (Agilent Technologies) were used for dataanalysis. The same parameters mentioned above wereemployed for expression analysis using GCOS. UsingGeneSpring, three sodium hypochlorite-treated samplereplicates, three hydrogen peroxide-treated replicates, andthree peracetic acid-treated replicates, with exposure timesof 10 min each, were normalized to three untreated(control) sample replicates. As previously described (Smallet al. 2007), the lists of genes with present/marginal callsfrom 50% or more of the replicates were created for eachsample set (three untreated control samples, three sodiumhypochlorite-treated samples, three hydrogen peroxide-treated samples, and three peracetic acid-treated samples).A master list was then created by merging the four genelists. A one-way ANOVA (p value≤0.05) was used todetermine statistically significant gene expression changeswithin this master list. Fold changes were calculated as theratios between the signal averages of three untreated(control) and the signal averages of each of thedisinfectant-treated (sodium hypochlorite (2.5 mM; Janget al. 2009a), hydrogen peroxide (0.5 mM; Jang et al.2009b), and peracetic acid (0.1 mM)) cultures.

Real-time PCR analysis: toxicogenomic responseof M. bovis BCG to peracetic acid

Quantitative real-time PCR on nine randomly selectedgenes was carried out in order to validate the transcriptlevels obtained by the microarray experiments. Primersequences and genes used for PCR analysis are listed inTable 1. The M. bovis BCG 16S recombinant RNA (rRNA)housekeeping gene was used as an internal control in thePCR reactions. The iCycler iQ PCR system, the iScriptcDNA synthesis kit, and the IQ SYBR Green Supermix(BioRad Laboratories, Inc., Hercules, CA, USA) were usedto perform the real-time PCR reactions. For each gene,three biological replicates with three technical replicateseach were employed. The conditions used for PCRreactions were 3 min at 95.0°C followed by 40 cycles of10 s at 95.0°C, 30 s at 55.0°C, and 20 s at 72.0°C. PCRefficiencies were determined from standard curve slopes inthe iCycler software v.3.1. Assessment of PCR specificitywas carried out using melt-curve analysis. Single primer-specific melting temperatures were obtained from melt-curve analysis. Changes in the expression of genes relative


to the 16S rRNA gene were used to quantify transcript levelchanges. Data on Table 1 indicate that our microarrayresults were in agreement with real-time PCR results.

Results

Growth inhibition of M. bovis BCG by peracetic acid

In order to determine a suitable sublethal concentration ofperacetic acid that will produce strong growth inhibition,M. bovis BCG was exposed to four concentrations ofperacetic acid (0.05, 0.1, 0.2, and 0.5 mM), and growthinhibition was monitored by the changes in the amounts ofATP in picomoles produced 10 min intervals for 1 h. InFig. 1, the highest concentration of peracetic acid used(0.5 mM) produced a drastic growth inhibition. Therefore, alower concentration of 0.1 mM was selected as the testconcentration to observe the sublethal effects of peraceticacid on M. bovis BCG.

Changes in the transcriptional profiles of M. bovis BCGin response to peracetic acid

Three microarray replicates were used in the absence(control) and in the peracetic acid-exposed (experimental)group. Transcriptome time course effects were observed

after 10 and 20 min of exposure to 0.1 mM peracetic acid.Determination of significant changes in transcription inresponse to peracetic acid was based on the followingcriteria: (1) the p value for a Mann–Whitney t test <0.05,(2) a ≥2-fold change in transcript level, and (3) a geneshould have a present or marginal call (Affymetrix, Inc.)from 50% or more replicates on both experimental andcontrol replicate sets. After a one-way ANOVA, 1,740 out ofthe 5,412 genes that make up theM. bovis BCG genome werefound to be statistically significant. Further analysis of thesegenes revealed that a total of 277 were upregulated ≥2-foldor downregulated ≤2-fold after 10 and 20 min. All datafrom this study have been deposited in the NationalCenter for Biotechnology information (NCBI) geneExpression Omnibus and can be accessed through theGEO series accession number GSE 15023.

Functional classification of upregulated and downregulatedgenes in M. bovis BCG in response to peracetic acidtreatment

The 277 statistically significant genes were classified basedon the COG functional categories specified by the NCBI.One hundred sixty-six genes were classified as “functionunknown”, “intergenic regions”, “hypothetical”, “generalfunction prediction only”, and “unclassified”. These geneshave not been included in Figs. 2 and 3. Figure 2 illustrates

Table 1 Transcript level comparison of M. bovis BCG genes between real-time PCR and microarray analyses

Gene mRNA levelchange withmicroarraya

mRNA levelchange with real-time PCRb

Forward primer sequence (5′–3′) Reverse primer sequence (5′–3′)

Fold change Fold change

10 min 20 min 10 min 20 min

BCG_1697 2.39 2.62 3.48 2.22 ATCAACGTCACCAAACGTTCGCC TTCGGTGTAGGCGTAGATGTCCTT

BCG_1698 2.79 2.81 3.18 2.07 TAGCTCGATCATGCCGCAGAAGAA AATCGAACACCGGCTCCTTGTCTT

BCG_2395c 2.07 2.14 2.76 2.07 ACTGGACTTGCGTAACCGACTCAA ACGCGAAATGTCCACTTTCTGTGC

BCG_1947c 2.19 2.41 3.1 2.32 AACATCAAAGTGTCCTTCGCCGAC GCAAAGGATTCCACGTCGGTTTGT

BCG_3156c 2.09 4.5 ATTGAACTGTGCCGCGATCTGTTG GCCAACTCCATTCCCTTGATGTCT

BCG_3155c 2.13 3.56 ATCCTGAAGTGCAGCAACGACTCT ACAATGGACCCACGAATTGAACGC

BCG_1249 −2.18 −2.15 TATGGCAGTGGGTGGGTACCAATA TTCTGGCCCTGGTAATCGAATGCT

BCG_2180c 2.03 3.32 GCAATTGGAAGAGGCCAGGAAGAA GGCATCTGGCTTGTTGTTCAGCTT

BCG_0299c −2.57 −2.12 −2.38 −3.45 AGTATCGCATCGAGCTGAACACCA TGCGAAAGAGAACCCGATGAACGA

16S rRNAc 1.00 1.00 1.00 1.00 TGC AAG TCG AAC GGA AAG GTCTCT

AAG ACA TGC ATC CCG TGG TCCTAT

a The microarray results are the mean of three replicates of each geneb The real-time PCR results are the mean of three biological replicates with three technical replicates for each genec Internal control: 16S rRNA


the grouping of 111 up- and downregulated genes at 10 and20 min into different functional classes and the totalnumber of genes in each class.

In Fig. 2, the functional classes of “coenzyme metabo-lism” and “inorganic ion transport and metabolism”contained more downregulated genes at 20 min comparedto 10 min. The functional classes of “amino acid transportand metabolism”, “DNA replication, recombination, andrepair”, “lipid metabolism”, and “signal transductionmechanisms” contained more upregulated genes at 10 mincompared to 20 min.

Grouping of functionally classified up- and downregulatedgenes inM. bovis BCG in response to peracetic acid treatment

The 111 up- and downregulated genes were placed in 6groups based on their transcription directions. Figure 3illustrates the six groups and the total number of genes ineach group. Group I contains genes that were upregulatedafter 10 and 20 min. Group II is made up of genes that wereupregulated after 10 min only. Group III contains genes thatwere downregulated only upon 10 min of peracetic acidtreatment. Group IV contains genes that were upregulated

after 20 min only. Group V is made up of genes that weredownregulated only after 20 min. Group VI contains genesthat were downregulated after both 10 and 20 min.

Changes in the transcriptional profiles of M. bovis BCGin response to sodium hypochlorite, hydrogen peroxide,and peracetic acid

Of the 5,412 genes represented in the M. bovis BCG customarray, 4,860 genes passed the present/marginal call from thefour sample sets (three untreated control samples, threesodium hypochlorite-treated samples, three hydrogenperoxide-treated samples, and three peracetic acid-treatedsamples) to form a master list. Based on the one-wayANOVA, 2,090, 2,069, and 1,973 genes in the sodiumhypochlorite, hydrogen peroxide, and peracetic acid samplesets, respectively, were statistically significant. When foldchange analysis was carried out, 84 genes in the sodiumhypochlorite-treated samples showed a 2-fold up- ordownregulation in expression compared to the controlsamples. Fifteen genes in the hydrogen peroxide-treatedsamples showed a 2-fold up- or downregulation inexpression compared to the control samples, and withinthe peracetic acid-treated samples, 290 genes were 2-foldup- or downregulated compared to the controls.

Venn diagram regions

A Venn diagram which shows the unions and intersectionsof the three disinfectants was constructed (Fig. 4). Fifty-twogenes were upregulated and five genes were downregulatedexclusively in response to sodium hypochlorite (region 1).Six genes were upregulated and no genes were downregulatedexclusively in response to hydrogen peroxide (region 2). Onehundred seventy genes were upregulated and 96 genes weredownregulated exclusively in response to peracetic acid(region 3). There were six upregulated genes and no down-regulated genes in common between sodium hypochlorite andhydrogen peroxide (region 4). Twenty upregulated genes andone downregulated gene were common between sodiumhypochlorite and peracetic acid (region 5). There werethree upregulated and no downregulated genes incommon between hydrogen peroxide and peracetic acid(region 6). No genes were found in common to all thethree disinfectants (region 7).

Heat map analysis of changes in the transcriptional profilesof M. bovis BCG in response to sodium hypochlorite,hydrogen peroxide, and peracetic acid

A heat map analysis illustrating the changes in geneexpression in the control samples and experimental samples

Fig. 1 Growth Inhibition of M. bovis BCG by peracetic acid over60 min. ATP measurements in picomoles were monitored in 10-minintervals. The peracetic acid concentrations were as follows: control withwater (filled square), 0.05 mM (filled triangle), 0.1 mM (inverted filledtriangle), 0.2 mM (filled diamond), and 0.5 mM (filled circle). Each datapoint was determined from the average of three separate experiments andthe error bars represent the standard deviations obtained


Fig. 3 Classification of significantly regulated 111 genes into 6groups based on their transcription directions after 10 and 20 min ofexposure to 0.1 mM peracetic acid. Filled bars indicate upregulationat either or both treatment times. Empty bars indicate downregulationat either one or both treatment times. Group I is made up of genesupregulated after both exposure times. Group II contains genesupregulated at 10 min, with no significant changes after 20 min of

exposure. Group III consists of genes downregulated after 10 min,with no significant changes upon 20 min of treatment. Group IV ismade up of genes that were upregulated in response to 20 min oftreatment. Group V is made up of genes that were downregulated upon20 min of treatment. Group VI is made up of genes that weredownregulated upon both treatment times

Fig. 2 Functional classificationof statistically significantupregulated (filled bars) anddownregulated (empty bars)genes after 10 and 20 min ofexposure to 0.1 mM peraceticacid. The numbers inparentheses indicate the totalnumber of genes for eachfunctional class in both groups(a total of 111 genes)


for sodium hypochlorite, hydrogen peroxide, and peraceticacid treatment was performed. Visual inspection of the heatmap indicates that sodium hypochlorite and peracetic acidtreatments led to more changes in gene expression (up- anddownregulation of genes) compared to hydrogen peroxidetreatment (Fig. 5).

Discussion

Toxicogenomic response of M. bovis BCG to peracetic acid

All of the genes discussed in this report are in theSupplementary material. However, for clarity and tofacilitate the reading of this report, the genes discussedbelow in the six groups are indicated in Table 2.

Group I: genes upregulated after 10 and 20 min of exposureto peracetic acid

This class contained eight genes relating to arginine biosyn-thesis namely BCG_1691–1698 (argC, argI, argB, argD andargF, argG and argH, and argR). All the genes involved inthe arginine biosynthetic pathway are essential for optimalgrowth of both M. tuberculosis and M. bovis BCG (Sassetti

et al. 2003). The upregulation of arginine biosynthesis in thisstudy corroborates the fact that arginine is necessary for M.bovis BCG growth but also points to the possibility thatarginine biosynthesis in M. bovis BCG may play a role in itsadaptation to peracetic acid-induced oxidative stress.

The polyketide synthase-associated gene, papA1(BCG_3887c) which encodes a probable acyltransferasewas upregulated after both treatment times. PapA1 isrequired for the biosynthesis of sulfolipid-1, which is amajor glycolipid of the M. tuberculosis cell wall and issuspected to be involved in virulence (Bhatt et al. 2007). Asecond polyketide synthase gene mbtD gene (BCG_2395c)was also upregulated in this group. MbtD encodes apolyketide synthase that is involved in the biosynthesis ofmycobactins which are salicylic acid-derived siderophores,important in mycobacterial iron acquisition (Barclay andRatledge 1983; LaMarca et al. 2004; Quadri et al. 1998;Snow 1970). Iron is essential in mycobacteria as a cofactorfor enzymes catalyzing redox reactions and for othercellular functions (Rodriguez and Smith 2006). In M.tuberculosis, mycobactins are essential for virulence andinfection maintenance (Neres et al. 2008; Rodriguez andSmith 2006). Mycobactins may also function as temporaryreservoirs of iron, potentially mediating the formation ofreactive oxygen species (De Voss et al. 2000; Snow 1970;Vergne et al. 2000). The upregulation of these virulence-associated genes points to the possibility that thepathogenesis of M. bovis BCG is induced in response toperacetic acid treatment. Similar results showing theupregulation of virulence have been reported in Pseudo-monas aeruginosa and S. aureus treated with differentantimicrobials (Chang et al. 2006a, b; Jang et al. 2008).

The catalase-peroxidase-peroxynitritase T (katG) genewas also upregulated after both treatment times. KatG is ahallmark anti-oxidative stress enzyme produced in pathogenicmycobacteria against reactive oxygen metabolites (Heym etal. 1993; Milano et al. 2001; Sherman et al. 1995). KatG isalso implicated as a virulence factor of M. tuberculosis basedon both guinea pig and mouse models (Li et al. 1998;Wilson et al. 1995). Iron regulation and oxidative stress areintricately connected (Milano et al. 2001; Zheng and Storz2000), with iron mediating the detrimental cytotoxic effectsof reactive oxygen species (Zheng and Storz 2000) and alsofunctioning as an essential cofactor of enzymes (Ernst et al.2005). The upregulation of both iron acquisition/virulenceand oxidative stress response genes in this study suggeststhat these processes are all involved in the adaptive responseof M. bovis BCG to peracetic acid treatment.

BCG_1255 (PE13) and BCG_3040c (PPE46) whichbelong to the PE/PPE families of genes were upregulatedafter both 10 and 20 min of peracetic acid exposure. ThePE/PPE families of genes constitute approximately 10% ofthe genome of M. tuberculosis (Tundup et al. 2006; Voskuil

Region 1NaOCl52 upregulated5 downregulated

Region 5

Region 2Region 4NaOCl 6 upregulatedH2O2

H2O2

H2O2

0 downregulated6 upregulated0 downregulated

Region 7Region5 NaOCl

Region 6 CH3CO3H

CH3CO3H

CH3CO3H

H2O2CH3CO3H0 genes3 upregulatedNaOCl0 downregulated20 upregulated

1 downregulated

Region 3

170genes upregulated96 genes downregulated

Fig. 4 Venn diagram showing intersections among the genes up- anddownregulated in M. bovis BCG in response to sodium hypochlorite,peracetic acid, and hydrogen peroxide treatment. Designated regionsare as follows: sodium hypochlorite is exclusively in region 1,hydrogen peroxide is exclusively in region 2, peracetic acid isexclusively in region 3, the intersection between sodium hypochloriteand hydrogen peroxide is in region 4, the intersection between sodiumhypochlorite and peracetic acid is in region 5, the intersection betweenhydrogen peroxide and peracetic acid is in region 6, and theintersection of all three antimicrobials is in region 7. The genes inthe regions represented in this diagram are listed in Table 3


et al. 2004) and are suggested to be cell wall proteins thatcould provide a diverse antigenic profile and affectimmunity (Voskuil et al. 2004). Studies have shown thatproteins belonging to these families may contribute toimmunity if included in a tuberculosis vaccine (Chaitra etal. 2008; Tundup et al. 2008). A recent study also indicatedthat PPE proteins may play a role in the transport ofantimicrobials across the M. bovis BCG outer membrane(Danilchanka et al. 2008). These results suggest that inaddition to the already reported functions for the PE/PPEgenes of mycobacteria, they may also play a role in theresponse to oxidative damage.

Group II: genes upregulated only upon 10 min of exposureto peracetic acid

Group II of Table 2 indicates the two genes of interest in thisgroup: BCG_3156c (devR) and BCG_3155c (devS). ThedevR–devS genes code for a response regulator, DevR and ahistidine sensor kinase, DevS, respectively, that manifestphosphorylation characteristics typical of two-componentsignal transduction systems (Saini et al. 2004). The DevR–DevS system regulates the genetic response ofM. tuberculosisto hypoxia and nitric oxide exposure (Bagchi et al. 2005),

both conditions which are likely to prevail during latenttuberculosis infections (Nathan and Shiloh 2000; Wayne andSohaskey 2001). The expression of devR–devS is alsoupregulated in M. bovis BCG grown in low oxygen environ-ments (Boon et al. 2001). Additionally, devR has beenimplicated in M. tuberculosis virulence in a guinea pig model,suggesting that it plays a critical and regulatory role in theadaptation and survival of M. tuberculosis within host tissues(Bagchi et al. 2005). Considering that M. bovis BCG in thisstudy was grown under aerobic conditions, the upregulationof the devR–devS system may occur in response to theeffects of oxidative stress due to the generation of reactiveoxygen species from peracetic acid treatment. Therefore,the upregulation of the devR–devS signal transductionsystem after 10 min with a return to normal transcriptionlevels after 20 min suggests that it may play a role in theearly protective response of M. bovis BCG to peraceticacid-induced oxidative stress.

Group III: genes downregulated only upon 10 minof exposure to peracetic acid

The glbN gene (BCG_1594c) was downregulated after10 min but returned to normal transcription levels after

Fig. 5 A heat map illustratingthe changes in gene expressionin control and experimentalsamples of M. bovis BCGtreated with sodium hypochlo-rite, hydrogen peroxide, andperacetic acid. Lanes 1, 2, and 3represent the control samples forexperiments with sodiumhypochlorite, hydrogenperoxide, and peracetic acidrespectively as treatments.Lanes 3, 4, and 5 represent theexperimental samples after10 min of exposure to 2.5 mMsodium hypochlorite, 0.5 mMhydrogen peroxide, and 0.1 mMperacetic acid, respectively.Upregulated genes are shown inred while downregulated genesare shown in green


Tab

le2

Listof

sign

ificantly

up-or

downregulated

M.bo

visBCG

genesin

respon

seto

peracetic

acid

treatm

entthat

arediscussedin

thisrepo

rt

Affym

etrixprob

eID

ORFno

.10

min

a20

min

aDescriptio

nSym

bol

Functionalclass

Fold

change

bPvalue

Fold

change

bPvalue

GroupI:

upregu

lation

(10min)–upregu

lation

(20min)

MBOV07

04S0000

1683_at

BCG_169

72.39

0.0018

2.62

0.00

18Putativeargininosuccinatesynthase

argG

argG

Aminoacid

transportandmetabolism

MBOV0704S00001680_at

BCG_1694

2.49

0.000245

2.75

0.000245

Putativeacetylornithineam

inotransferase

argD

argD

Aminoacid


MBOV07

04S0000

1682_at

BCG_169

62.62

0.0010

72.58

0.00

107

Putativearginine

repressorargR

argR

Transcriptio

n

MBOV07

04S0000

1681_at

BCG_169

52.65

0.0005

132.90

0.00

0513

Putativeornithinecarbam

oyltransferase,

anabolic

ArgF

argF

Aminoacid


MBOV07

04S0000

1679_at

BCG_169

32.75

0.0005

292.87

0.00

0529

Putativeacetylglutam

atekinase

argB

argB

Aminoacid


MBOV0704S00001678_at

BCG_1692

2.77

0.000218

2.91

0.000218

Putativeglutam

ateN-acetyltransferaseargJ

argJ

Aminoacid


MBOV07

04S0000

1684_at

BCG_169

82.79

0.0112

2.81

0.0112

PutativeargininosuccinatelyaseargH

argH

Aminoacid


MBOV07

04S0000

1677_at

BCG_169

12.33

0.0035

22.55

0.00

352

PutativeN-acetyl-gamma-glutam

yl-phoshate

reductaseargC

argC

Aminoacid


MBOV07

04S0000

2377_at

BCG_239

5c2.07

0.0443

2.14

0.04

43Polyketidesynthetase

mbtD

mbtD

Secondary

metabolitesbiosynthesis,

transport,andcatabolism

MBOV0704S00001931_at

BCG_1947c

2.19

0.000291

2.41

0.000291

Catalase-peroxidase-peroxynitritase

TkatG

katG

Inorganiciontransportandmetabolism

MBOV0704S00003859_at

BCG_3887c

2.34

0.000352

2.48

0.000352

Putativepolyketid

esynthase-associatedprotein

papA

1pa

pA1

Secondary

metabolitesbiosynthesis,

transport,andcatabolism

MBOV07

04S0000

1241_at

BCG_125

52.76

0.0072

72.58

0.00

727

PEfamily

protein

PE13

MBOV07

04S0000

3017_at

BCG_304

0c2.36

0.0221

2.40

0.02

21PPEfamily

protein

PPE46

GroupII:upregu

lation

(10min)–nochan

ge(20min)

MBOV07

04S0000

3133_at

BCG_315

6c2.09

0.0066

7Tw

o-compo

nent

transcriptionalregulatory

protein

devR

(probablyluxR

/uhpA

family

)devR

Signaltransductio

nmechanism

s

MBOV0704S00003132_at

BCG_3155c

2.13

0.0154

Two-component

sensor

histidinekinase

devS

devS

Signaltransductio

nmechanism

s

GroupIII:

dow

nregu

lation

(10min)–nochan

ge(20min)

MBOV07

04S0000

1580_at

BCG_159

4c−2

.38

0.0041

6Putativehemog

lobinglbN

glbN

General

functio

npredictio

nonly

MBOV07

04S0000

1880_at

BCG_189

6−2

.20

0.000974

Alanine-andproline-rich

secreted

proteinapa

apa

MBOV07

04S0000

1235_at

BCG_124

9−2

.18

0.0341

Putativepyrroline-5-carboxylatedehydrogenase

rocA

rocA

Energyprod

uctio

nandconversion

GroupIV

:nochan

ge(10min)–upregu

lation

(20min)

MBOV0704S00002162_at

BCG_2180c

2.03

0.0158

Putativepenicillin-bindingmem

brane

proteinpb

pBpb

pBCellenvelope

biog

enesis,ou

ter

mem

brane

MBOV07

04S0000

2781_at

BCG_280

2c2.04

0.00

0264

Putativelip

oprotein

lppU

lppU

MBOV07

04S0000

2122_at

BCG_214

02.09

0.01

68PPEfamily

protein

PPE37

GroupV:nochan

ge(10min)–dow

nregulation

(20min)

MBOV07

04S0000

3622_at

BCG_365

0−2

.27

4.37E−0

5DNA

repairproteinradA

radA

MBOV07

04S0000

3296_at

BCG_331

9c−2

.07

0.02

19Putative

L-lysine-epsilonam

inotransferase

lat

lat

Aminoacid


GroupVI:

dow

nregu

lation

(10min)–dow

nregu

lation

(20min)

MBOV07

04S0000

0291_at

BCG_029

9c−2

.57

0.0111

−2.12

0.0111

Putativeintegral

mem

branenitrite

extrusionprotein

narK

3Inorganiciontransportandmetabolism


20 min (Table 2). This gene encodes a truncated hemoglo-bin, designated trHbN whose sequence is identical in bothM. tuberculosis and M. bovis (Wittenberg et al. 2002).Previous studies have suggested that trHbN plays a role inthe detoxification of reactive nitric oxide generated byactivated macrophages during physiological studies of M.bovis BCG and also during M. tuberculosis infections(Couture et al. 1999; Pawaria et al. 2008).

A second downregulated gene in this group was the apagene which encodes an alanine- and proline-rich secretedprotein (Table 2). The Apa molecules secreted by M. bovisBCG function as major immunodominant antigens (Horn etal. 1999). The addition of a poxvirus recombinant boostexpressing an Apa protein of M. tuberculosis to a DNAvaccine led to a significant reduction of mycobacterialcounts in the spleens of immunized guinea pigs comparableto the reduction obtained by the BCG vaccine (Kumar et al.2003).

Another downregulated gene in this group wasBCG_1249 (rocA) (Table 2). The rocA gene has beenproposed to play a role in the adaptation of Mycobacteriumavium subsp. paratuberculosis to its niche and theutilization of carbon sources within (Hughes et al. 2007).

Group IV: genes upregulated only upon 20 min of exposureto peracetic acid

In this group, BCG_2180c which encodes a putativepenicillin-binding membrane protein PbpB was upregulatedafter 20min of exposure to peracetic acid (Table 2). Penicillin-binding proteins are serine acyl transferases involved in thefinal stages of peptidoglycan synthesis and contribute to cellwall expansion, cell shape maintenance, septum formation,and cell division (Goffin and Ghuysen 2002; Popham andYoung 2003). A recent study reported that the remodeling ofthe peptidoglycan network of M. tuberculosis may beinvolved in the adaptive response to the treatment oftuberculosis infections with a combination of antibiotics(Lavollay et al. 2008). In addition, another study reporteda possible connection between peptidoglycan biosynthesisand oxidative stress defense in Streptococcus thermophilus(Thibessard et al. 2002). In the aforementioned study, thepbp2 gene (which is reported to be implicated inpeptidoglycan biosynthesis during the process of cellelongation) is shown to be involved in the response tohydrogen peroxide-induced oxidative stress.

A second upregulated gene in this group wasBCG_2802c which encodes a putative lipoprotein. Myco-bacterial lipoproteins are usually cell surface-associated andare important for the formation of the cell envelope andsensing of and protection from environmental stress, andthey play a role in host pathogen reactions (Rezwan et al.2007). In addition, lipoprotein metabolism has beenT

able

2(con

tinued)

Affym

etrixprob

eID

ORFno

.10

min

a20

min

aDescriptio

nSym

bol

Functionalclass

Fold

change

bPvalue

Fold

change

bPvalue

narK

3MBOV07

04S0000

3084

_at

BCG_310

7c−2

.54

0.00

35−2

.05

0.0035

Virulence-regulatingtranscriptionalregulatorvirS

(araC/xylSfamily

)virS

Transcriptio

n

MBOV07

04S0000

1993

_at

BCG_200

9c−2

.19

0.00

0331

−2.05

0.000331

Putativemetal

catio

ntransporterP-typeatpase

GctpG

ctpG

Inorganiciontransportandmetabolism

MBOV07

04S0000

1671

_at

BCG_168

5−2

.07

0.00

683

−2.08

0.0068

3PEfamily

protein

PE17

MBOV07

04S0000

1662

_at

BCG_167

6−2

.03

0.00

0509

−2.01

0.0005

09ExcinucleaseABC,subunitauv

rAuvrA

DNA

replication,

recombinatio

n,and

repair

MBOV07

04S0000

3049

_at

BCG_307

2c−2

.15

0.00

128

−2.16

0.00128

Ribonucleoside-diphosphatereductasesubunitbeta

nrdF

2nrdF

2Nucleotidetransportandmetabolism

aThe

microarrayresults

arethemeanof

threereplicates

ofeach

gene

bThe

fold

change

isapo

sitiv

enu

mberwhentheexpression

levelintheexperimentincreased

comparedto

thecontroland

isanegativ

enu


levelintheexperimentd

ecreased

compared

tothecontrol


established as a major virulence determinant for tuberculo-sis (Sander et al. 2004).

The upregulation of these cell wall-associated genes inresponse to peracetic acid treatment may be indicative ofcell wall modification as a protective strategy againstperacetic acid treatment. In addition, lipoprotein-associated virulence further supports the results in group Iwhich indicate that virulence mechanisms in M. bovis BCGmay contribute to the adaptive response to peracetic acidexposure.

Another gene of the PPE family BCG_2140 (PPE37)was upregulated only upon 20 min of exposure to peraceticacid (Table 2). Two genes belonging to the PE/PPE familyof genes were upregulated after both treatment times (seegroup I). Currently, the functions of all PPE proteins are notknown. These results support the fact that these genes elicitdiversified metabolic functions.

Group V: genes downregulated only upon 20 minof exposure to peracetic acid

The radA gene (BCG_3650) which is involved in DNArepair was downregulated after 20 min (Table 2). Thetranscription of the radA gene was upregulated in M.tuberculosis grown in a simulated phagosomal environment(Schnappinger et al. 2003). The expression of radA in M.tuberculosis also increased following induced DNA dam-age (Rand et al. 2003). These results suggest that peraceticacid on M. bovis BCG may include the inhibition of someDNA repair genes.

A second downregulated gene in this group was the latgene (BCG_3319c) which encodes a putative L-lysine-epsilon aminotransferase. Expression of the LAT proteinwas upregulated approximately 40-fold during the latentphase of M. tuberculosis, suggesting that it plays asignificant role for survival (Tripathi and Ramachandran2006). As such, the downregulation of the latA gene maycontribute to the peracetic acid-induced growth inhibitionobserved in this study.

Group VI: genes downregulated after 10 and 20 minof exposure to peracetic acid

The narK3 gene (BCG_0299c), which codes for a putativeintegral membrane nitrite extrusion protein, was down-regulated after both treatment times (Table 2). TheEscherichia coli narK gene is implicated in nitrate uptakeor nitrite excretion (DeMoss and Hsu 1991; Noji et al.1989). The M. tuberculosis narK1 through narK3 and narUare homologous to the E. coli narK and narU (Cole et al.1998). In M. tuberculosis, nitrite production is upregulatedduring anaerobic conditions, and the transcription of thenitrite transport gene, narK2, is also upregulated (Sohaskey

and Wayne 2003). However, in M. bovis, only low levels ofnitrite were produced, and this was not induced by hypoxia(Sohaskey and Wayne 2003). The downregulation of narK3gene in this study suggests that when M. bovis BCG issubjected to oxidative stress, anaerobic metabolism wherenitrate is used as a terminal electron acceptor and isconverted to nitrite is not favored.

Another downregulated gene in this group wasBCG_3107c, virS. The virS gene encodes a transcriptionalregulator which belongs to the AraC family and is involvedin the regulation of pathogenesis of M. tuberculosis (Guptaet al. 1999; Gupta and Tyagi 1993). In a recent study, thevirulence regulator virS was upregulated during thereactivation phase of tuberculosis and was suggested to beone of the master regulators in the reactivation oftuberculosis (Talaat et al. 2007). The downregulation ofvirS after both 10 and 20 min contrasts the results obtainedin group I which support the upregulation of virulence inresponse to peracetic acid treatment. This suggests that thevirulence genes of M. bovis BCG may elicit differentmetabolic roles, thus are differentially affected by peraceticacid treatment.

A third downregulated gene in this group was ctpG,which encodes a putative metal cation-transporting P-typeATPase. In a previous study, ctpG appeared to be inducedby low iron and it was theorized that ctpG may transportiron (De Voss et al. 2000). The downregulation of ctpG inthis study, therefore, supports the results in group 1 thatindicate that peracetic acid treatment may elicit theregulation of intracellular iron levels to ensure growth andsurvival but also to combat the effect of oxidant-induceddamage.

BCG_1685 (PE 17) which belongs to the PE/PPE familyof genes was downregulated after both 10 and 20 min ofexposure to peracetic acid. This further showed that the PE/PPE families of genes elicit diversified metabolic functions.

The uvrA gene (BCG_1676) was downregulated ap-proximately 2-fold after both treatment times. UvrA iscritical to the nucleotide excision repair (NER) processwhich is used by cells to repair a wide range of DNAlesions (Croteau et al. 2006). During the NER process,UvrA initially recognizes distortions caused by damage inDNA and then transfers the damaged DNA to UvrB whichmakes a more detailed evaluation of the nature of damage(Croteau et al. 2006, 2008; Zou et al. 1998). The down-regulation of the uvrA gene after both treatment timessuggests that peracetic acid may affect DNA damage/repairsystems in M. bovis BCG. The downregulation of nrdF2gene (BCG_3072c) further supports this theory. The nrdF2encodes the ribonucleoside-diphosphate reductase subunitbeta. Ribonucleotide reductases are critical to all living cellsbecause they provide deoxyribonucleotides for DNAsynthesis and repair (Mowa et al. 2009). In addition, both


Tab

le3

Listof

genesrepresentedin

theVenndiagram

byregion

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

Region1:

genesupregu

latedbysodium

hyp

ochlorite

only:52

genes

AFFX-BioB-M

_at

4.02

0.0132

E.coli/GEN=bioB

/DB_X

REF=gb:J04423.1/

NOTE=SIF

correspondingto

nucleotid

es2482–-

2739

ofgb

:J0442

3.1/DEF=E.coli7,8-diam

ino-

pelargonic

acid

(bioA),biotin

synthetase

(bioB),

7-keto-8-amino-pelargon

icacid

synthetase

(bioF),bioC

protein,

anddethiobiotin

synthetase

(bioD),completecds

MBOV0704S00000016_s_at

BCG_0019

5.37

0.00175

PutativethioredoxinreductasetrxB

2(TRXR)

trxB

2_2

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S00

000017

_s_at

BCG_0

020

5.28

0.0035

6Thioredox

intrxC

(TRX)(M

PT46)

trxC

_2Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S00

000171

_at

BCG_0

178

3.17

0.0006

64Hyp

othetical

protein

MBOV0704S00000356_at

BCG_0364

2.65

0.00123

Putativetranscriptionalregulatory

protein(possibly

arsR

family

)Inorganiciontransport

andmetabolism

MBOV07

04S00

000358

_at

BCG_0

366c

2.25

0.0063

7PutativecytochromeP45013

5A1cyp1

35A1

Secon

dary

metabolites

biosynthesis,

transport,and

catabo

lism

MBOV07

04S00

000362

_at

BCG_0

370

8.96

0.0002

45Putativedehydrog

enase/redu

ctase

General

functio

npredictio

non

lyMBOV07

04S00

000414

_at

BCG_0

422c

2.59

0.0033

Putativeendo

peptidaseATP-binding

protein(chain

b)clpB

clpB

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S00

000604

_at

BCG_0

614

2.12

0.0047

9Hyp

othetical

protein

General

functio

npredictio

non

lyMBOV07

04S00

001035

_at

BCG_1

046c

2.63

0.0106

Hyp

othetical

serine-richprotein

General

functio

npredictio

non

lyMBOV07

04S00

001095

_at

BCG_110

715

.21

0.0002

48Putativetranscriptionalrepressorprotein

Transcriptio

n

MBOV0704S00001096_at

BCG_1108

22.89

0.000118

Putativeoxidoreductase

General

functio

npredictio

non

lyMBOV0704S00001118_at

BCG_1130

2.15

0.00191

Putativetransm

embraneprotein

Functionunknow

n

MBOV07

04S00

001119

_at

BCG_113

13.03

0.0006

2Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

001267

_at

BCG_1

281

2.16

0.0357

AlternativeRNA

polymerasesigm

afactor

sigE

Fun

ctionun

know

n

MBOV07

04S00

001268

_at

BCG_1

282

2.02

0.0010

6Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

001518

_at

BCG_1

532

7.23

0.0002

08PutativethioredoxintrxB

1trxB

1Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S00

001519

_at

BCG_1

533

2.44

0.0024

5Putativeenoyl-CoA

hydrataseechA

12echA

12Lipid

metabolism

MBOV07

04S00

001566

_at

BCG_1

580c

3.62

0.0009

28Putativepolyketidesynthase-associatedproteinpapA

4pa

pA4

MBOV07

04S00

001697

_at

BCG_1

711c

3.49

0.0059

2Hyp

othetical

protein

Fun

ctionun

know

n


Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV0704S00001698_at

BCG_1712c

3.66

0.00212


protein

Inorganiciontransport

andmetabolism

MBOV07

04S0000

1763_at

BCG_177

72.26

0.00

102

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

1895_at

BCG_1911

4.15

0.00

167

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV0704S00002008_at

BCG_2024c

2.01

0.00493

Putativeferredoxin

fdxA

fdxA

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

2034_at

BCG_205

0c2.28

0.00

11HeatshockproteinhspX

hspX

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S0000

2053_at

BCG_206

92.31

0.02

04Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2055_at

BCG_207

1c2.06

0.00

624

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2056_at

BCG_207

2c2.09

0.00

187

Putativetransm

embraneprotein

MBOV07

04S0000

2122_at

BCG_214

02.62

0.00

144

PPEfamily

protein

Cellmotility

and

secretion

MBOV0704S00002394_at

BCG_2412c

2.29

0.00359

Putativesulfate-transportATP-binding

protein

ABCtransportercysA

1cysA

1Inorganiciontransport

andmetabolism

MBOV0704S00002395_at

BCG_2413c

2.40

0.00119

Putativesulfate-transportintegral

mem

brane

proteinABCtransportercysW

cysW


andmetabolism

MBOV07

04S0000

2397_at

BCG_241

5c2.17

0.00

074

Putativesulfate-bind

inglip

oprotein

subI

subI


andmetabolism

MBOV07

04S0000

2623_at

BCG_264

4c5.09

0.00

016

Putativetransm

embraneprotein

MBOV07

04S0000

2686_at

BCG_270

7c2.40

0.00

0641

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2691_at

BCG_271

2c2.39

0.00

0585

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2702_at

BCG_272

32.38

0.02

48RNA

polymerasesigm

afactor

sigB

Transcriptio

n

MBOV07

04S0000

2739_at

BCG_276

0c2.09

0.0119

Conserved

35kD

aalanine-rich

protein

Transcriptio

nSignal

transductio

nmechanism

s

MBOV0704S00002740_at

BCG_2761c

2.23

0.0022


protein

MBOV0704S00003054_at

BCG_3077c

2.67

0.00906

Putativeglutaredoxin

electron

transportcomponent

ofnrdE

F(glutaredoxin-lik

eprotein)

nrdH

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV0704S00003175_at

BCG_3198

5.37

0.000665

Putativeshort-chaindehydrogenase/reductase

General

functio

npredictio

nonly

MBOV07

04S0000

3209_at

BCG_323

2c3.78

0.00

142

Putativemolybdenu

mcofactor

biosynthesisprotein

moeB1

moeB1

Coenzym

emetabolism

MBOV0704S00003255_at

BCG_3278c

2.32

0.0366


protein

(probablytetR

family

)Transcriptio

n

MBOV07

04S0000

3256_at

BCG_327

9c2.67

0.02

03Putativerubredox

inrubB

rubB

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

3257_at

BCG_328

0c2.18

0.01

57Putativerubredox

inrubA

rubA

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

3941_s_at

BCG_397

15.08

0.00

128

PutativethioredoxinreductasetrxB

2trxB

2_2

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S0000

3942_s_at

BCG_397

24.84

0.00

67Thioredoxin

trxC

trxC

_2Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S0000

3964_at

3.21

0.00

477

Intergenic

region

21218–21

326

MBOV07

04S0000

4348_at

4.81

7.24

E−0

5Intergenic

region

120427

3–12

0443

0


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S00

004420

_at

3.02

0.0346

Intergenic

region

139490

7–13

9506

3

MBOV07

04S00

004638

_at

2.02

0.0035

9Intergenic

region

212578

4–21

2629

8

MBOV07

04S00

004919

_at

2.07

0.0144

Intergenic

region

2977118–29

7724

9

MBOV07

04S00

005341

_at

3.51

0.0177

Intergenic

region

436607

3–43

6618

1

Region1:

genesdow

nregu

latedbysodium

hyp

ochlorite

only:5genes

MBOV07

04S00

000528

_at

BCG_053

6−2

.00

0.05


protein

(probablygn

tRfamily

)Transcriptio

n

MBOV07

04S00

000864

_at

BCG_087

6c−2

.05

0.0135


protein

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S00

001594

_at

BCG_160

8−2

.64

0.0048

3Putativemem

braneproteinmmpS

6

MBOV07

04S00

002000

_at

BCG_201

6c−2

.16

0.00833

Putativeintegral

mem

braneprotein

Aminoacid

transport

andmetabolism

MBOV07

04S00

004336

_at

−2.10

0.0244

Intergenic

region

1174

953–1175

048

Region2:

genesupregu

latedbyhyd

rogenperox

ideon

ly:6genes

MBOV07

04S00

001930

_at

BCG_194

6c3.82

6.75E−0

5Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

001932

_at

BCG_194

8c4.88

6.78E−0

5Ferricuptake

regulatio

nproteinfurA

furA


andmetabolism

MBOV07

04S00

002378

_at

BCG_239

6c4.46

4.22E−0

5Polyk

etidesynthetase

mbtC

mbtC

Secondary

metabolites

biosynthesis,

transport,and

catabolism

MBOV0704S00002379_at

BCG_2397c

3.65

0.000857

Phenyloxazolin

esynthase

mbtB

mbtB

Secondary

metabolites

biosynthesis,

transport,and

catabolism

MBOV0704S00002985_at

BCG_3008c

2.40

0.000441

Putative3-isopropylm

alatedehydratasesm

all

subunitleuD

leuD

Aminoacid

transport

andmetabolism

MBOV0704S00002986_at

BCG_3009c

3.39

0.00332

Putative3-isopropylm

alatedehydrataselarge

subunitleuC

leuC

Aminoacid

transport

andmetabolism

Region3:

genesupregu

latedbyperacetic

acid

only:17

0genes

MBOV07

04S00

000025

_s_at

BCG_002

8c2.20

0.0008

86Putativehemolysin

Fun

ctionun

know

n

MBOV07

04S00

000058

_at

BCG_006

22.09

0.0032

7Putativeremnant

ofatransposase

MBOV07

04S00

000058

_x_at

BCG_006

22.43

0.0174

Putativeremnant

ofatransposase

MBOV07

04S00

000080

_at

BCG_008

42.08

0.0004

17Putative30

sribo

somal

proteins6

rpsF

rpsF

General

functio

npredictio

nonly

MBOV07

04S00

000110

_at

BCG_0113

2.64

0.0151

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000111_at

BCG_0114

3.60

0.0005

12Putativetranscriptionalregulatory

protein

Transcriptio

n

MBOV0704S00000112_at

BCG_0115

4.10

0.00135


Energyproductio

nand

conversion

MBOV0704S00000113_at

BCG_0116

4.60

0.000108


Energyproductio

nand

Inorganicion


()

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

conversion

transportand

metabolism

MBOV07

04S0000

0114

_at

BCG_0117

2.52

0.01

79Putativeform

atehy

drog

enlyasehy

cD(FHL)

hycD

Energyprod

uctio

nand

conv

ersion

MBOV0704S00000117_at

BCG_0120

3.36

0.0269

Putativeform

atehydrogenasehycQ

(FHL)

hycQ

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

0125_at

BCG_012

8c3.33

0.01

44Hyp

othetical

protein

Functionun

know

n

MBOV0704S00000144_at

BCG_0147

2.11

0.0104

Putativedehydratase

Aminoacid

transport

andmetabolism

MBOV0704S00000261_at

BCG_0269

3.12

0.00162


protein

(probablytetR/acrRfamily

)Transcriptio

n

MBOV07

04S0000

0319_at

BCG_032

72.24

0.00

159

Hyp

othetical

proteinTB9.8

Functionun

know

n

MBOV07

04S0000

0381_at

BCG_038

92.46

0.00

349

Putativechaperon

eproteindn

aKdn

aKPosttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S0000

0382_at

BCG_039

02.76

0.00

229

PutativegrpE

protein(hsp-70cofactor)

grpE

Posttranslational

modification,

protein

turnov

er,chaperon

esMBOV07

04S0000

0606_at

BCG_061

6c2.25

0.00

378

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

0609_at

BCG_061

9c2.06

0.00

836

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

0703_at

BCG_071

42.24

0.01

67Hyp

othetical

protein

Functionun

know

n

MBOV0704S00000735_at

BCG_0746

2.02

0.00833

Putativedehydrogenase

Aminoacid

transport

andmetabolism

MBOV07

04S0000

0756_at

BCG_076

72.05

0.00

463

Putative30

Sribosomal

proteinS14

rpsN

1rpsN

1Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S0000

0757_at

BCG_076

82.18

0.00

31Putative30

Sribosomal

proteinS8rpsH

rpsH

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S0000

0762_at

BCG_077

32.10

0.00

479

Putative50

Sribosomal

proteinL15

rplO

rplO

Translatio

n,ribosomal

structureand

biog

enesis

MBOV0704S00001017_at

BCG_1028c

3.91

0.00265

Putativeacetyl-/propionyl-coacarboxylasesubunit

beta

accD

2accD

2Lipid

metabolism

MBOV07

04S0000

1018_at

BCG_102

9c2.63

0.00

0285

Putativeacyl-CoA

dehydrog

enasefadE

13fadE

13General

functio

npredictio

nonly

Function

unkn

own

MBOV07

04S0000

1019_at

BCG_103

0c2.98

7.95

E−0

5Hyp

othetical

protein

Functionun

know

n

MBOV0704S00001093_s_at

BCG_1105

2.62

0.0365

PutativeIS1081

transposase

DNAreplication,

recombination,and

repair

MBOV07

04S0000

1237_at

BCG_125

12.39

0.0117

PutativealternativeRNA

polymerasesigm

afactor

sigI′

sigI

Transcriptio

nGeneral

functio

npredictio

non

ly

MBOV07

04S0000

1241_at

BCG_125

52.76

0.00

878

PEfamily

protein

MBOV07

04S0000

1297_at

BCG_1311c

3.24

0.00

02Hyp

othetical

protein

Functionun

know

n

MBOV0704S00001312_at

BCG_1326c

2.29

0.000766


proteinem

bRSignaltransductio

nmechanism

sMBOV0704S00001316_at

BCG_1330c

2.03

0.0295

Hypothetical

secreted

protein

Functionunknow

n


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV0704S00001406_at

BCG_1420

2.87

0.0108


protein

General

functio

npredictio

nonly

MBOV07

04S00

001421

_at

BCG_143

5c2.05

0.0032

5Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

001422

_at

BCG_143

62.03

0.0212

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

001590

_at

BCG_160

42.72

0.023

Putativefumaratereductase[flavo

proteinsubunit]

frdA

frdA

Energyproductio

nand

conv

ersion

MBOV07

04S00

001663

_at

BCG_167

7c2.21

0.0181

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

001677

_at

BCG_169

12.33

1.97E−0

5PutativeN-acetyl-gamma-glutam

yl-phoshate

reductaseargC

argC

Aminoacid

transport

andmetabolism

MBOV07

04S00

001678

_at

BCG_169

22.77

3.66

E−0

5Putativeglutam

ateN-acetyltransferaseargJ

argJ

Aminoacid

transport

andmetabolism

MBOV0704S00001679_at

BCG_1693

2.75

0.000283

Putativeacetylglutam

atekinase

argB

argB

Aminoacid

transport

andmetabolism

MBOV07

04S00

001680

_at

BCG_169

42.49

4.31E−0

6Putativeacetylornithineam

inotransferase

argD

argD

Aminoacid

transport

andmetabolism

MBOV0704S00001681_at

BCG_1695

2.64

0.000105

Putativeornithinecarbam

oyltransferase,

anabolic

ArgF

argF

Aminoacid

transport

andmetabolism

MBOV07

04S00

001682

_at

BCG_169

62.62

9.47E−0

5Putativearginine

repressorargR

argR

Transcriptio

n

MBOV07

04S00

001683

_at

BCG_169

72.39

0.0005

98Putativeargininosuccinatesynthase

argG

MBOV07

04S00

001684

_at

BCG_169

82.79

0.0018

2PutativeargininosuccinatelyaseargH

argH

Aminoacid

transport

andmetabolism

MBOV07

04S00

001741

_at

BCG_175

52.12

0.0043

3Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

001832

_at

BCG_184

7c2.36

0.0102

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

001969

_at

BCG_198

62.01

0.0059

6Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002004

_at

BCG_202

0c4.06

0.0015

6Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002005

_at

BCG_202

1c2.62

0.0006

34Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002006

_at

BCG_202

2c2.42

0.0002

51Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002031

_at

BCG_204

7c2.80

0.0047

2Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002032

_at

BCG_204

8c3.78

0.023

Putativeph

osph

ofructok

inasepfkB

pfkB

Carbo

hydratetransport

andmetabolism

MBOV07

04S00

002061

_at

BCG_207

7c4.07

0.0159

Ribosom

alproteinL28

Translatio

n,ribo

somal

structure,

and

biog

enesis

MBOV07

04S00

002062

_at

BCG_207

82.09

0.0045

3Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002266

_at

BCG_228

4c2.11

0.0002

75Hyp

othetical

protein

Functionun

know

n

MBOV07

04S00

002280

_at

BCG_229

83.23

0.0001

26Hyp

othetical

protein

Functionun

know

n

MBOV0704S00002320_at

BCG_2338

3.52

0.0212

Putativesugar-transportintegral

mem

braneprotein

ABCTranspo

rter

uspB

Carbo

hydratetransport

andmetabolism

MBOV07

04S00

002544

_at

BCG_256

52.60

0.0134

Putativelip

oprotein

lppA

lppA

MBOV07

04S00

002629

_at

BCG_265

02.13

0.0034

3Hyp

othetical

proteinTB31.7

Functionun

know

n

MBOV07

04S00

002630

_at

BCG_265

1c4.06

0.0075

5Hyp

othetical

protein

Functionun

know

n


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV0704S00002658_at

BCG_2679

9.18

0.00461

Putativetransposaseforinsertionsequence

elem

ent

IS10

81DNA

replication,

recombinatio

n,and

repair

MBOV0704S00002658_s_at

BCG_2679

2.51

0.0313

Putativetransposaseforinsertionsequence

elem

ent

IS10

81DNA

replication,

recombinatio

n,and

repair

MBOV07

04S0000

2737_at

BCG_275

8c2.06

0.04

53Hyp

othetical

arginine-richprotein

MBOV07

04S0000

2757_at

BCG_277

8c2.10

0.00

0216

Hyp

othetical

protein

MBOV0704S00002759_at

BCG_2780c

2.07

0.000123

PutativedihydrofolatereductasedfrA

dfrA

Coenzym

emetabolism

MBOV07

04S0000

2784_at

BCG_280

52.18

0.00

344

Hyp

othetical

alanine-rich

protein

Celldivision

and

chromosom

epartitioning

MBOV07

04S0000

2988_at

BCG_3011c

2.04

0.01

02Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2997_at

BCG_302

0c2.91

0.03

93Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3017_at

BCG_304

0c2.36

0.03

14PPEfamily

protein

Cellmotility

and

secretion

MBOV07

04S0000

3021_at

BCG_304

4c3.60

0.04

27PPEfamily

protein

Cellmotility

and

secretion

MBOV0704S00003112_at

BCG_3135

2.86

0.0182

Putativepterin-4-alpha-carbinolaminedehydratase

moaB1

Coenzym

emetabolism

MBOV0704S00003117_s_at

BCG_3140

2.68

0.0181

Putativetransposase

DNA

replication,

recombinatio

n,and

repair

MBOV0704S00003122_at

BCG_3145

4.50

0.0115


protein

Signaltransductio

nmechanism

sMBOV0704S00003132_at

BCG_3155c

2.13

0.000929

Two-component

sensor

histidinekinase

devS

devS

Signaltransductio

nmechanism

sMBOV0704S00003133_at

BCG_3156c

2.09

0.00397

Two-component

transcriptionalregulatory

protein

devR

(probablyluxR

/uhpA

family

)devR

Signaltransductio

nmechanism

sTranscriptio

n

MBOV07

04S0000

3134_at

BCG_315

7c2.08

0.01

81Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3180_at

BCG_320

32.39

0.03

69Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3300_s_at

BCG_332

3c2.32

0.01

27Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3371_at

BCG_339

6c2.47

0.00

56Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3378_at

BCG_340

3c2.65

0.01

56Hyp

othetical

proline-rich

protein

Fun

ctionun

know

n

MBOV07

04S0000

3397_at

BCG_342

5c3.12

6.13E−0

7Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3424_at

BCG_345

2c2.21

0.00

0551

Putativepo

lyprenyl

synthetase

idsB

idsB

Coenzym

emetabolism

MBOV07

04S0000

3444_at

BCG_347

2c2.80

0.00

725

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3484_at

BCG_351

2c2.20

0.00

455

Hyp

othetical

alanine-

andvalin

e-rich

protein

MBOV0704S00003492_at

BCG_3520c

2.36

0.000802

PutativetRNA

pseudouridinesynthase

AtruA

truA

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S0000

3494_at

BCG_352

2c2.67

7.35

E−0

5PutativeDNA-directedRNA

polymerase(alpha

chain)

rpoA

rpoA

Transcriptio

n

MBOV07

04S0000

3495_at

BCG_352

3c2.10

0.00

116

Putative30

Sribo

somal

proteinS4rpsD

rpsD

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV0704S00003668_at

BCG_3696

5.04

0.0371

Putativetransposase

DNA

replication,


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

recombinatio

n,and

repair

MBOV07

04S00

003760

_at

BCG_3

787

6.72

0.0221

Putativeox

idoreductase

Fun

ctionun

know

n

MBOV07

04S00

003859

_at

BCG_3

887c

2.34

3.11E−0

6Putativepolyketid

esynthase-associatedprotein

papA

1pa

pA1

Secondary

metabolites

biosynthesis,

transport,and

catabo

lism

MBOV0704S00003950_s_at

BCG_3980c

2.08

0.0015

Putativehemolysin

Functionunknow

n

MBOV07

04S00

003968

_at

2.01

0.0028

6Intergenic

region

3119

2–31

718

MBOV07

04S00

003975

_at

2.03

0.0303

Intergenic

region

43225–43

379

MBOV07

04S00

003981

_at

7.14

0.0144

Intergenic

region

57091–57

242

MBOV07

04S00

004006

_at

2.96

0.0032

9Intergenic

region

1192

22–119

315

MBOV07

04S00

004068

_at

3.16

0.0075

6Intergenic

region

330684–330

896

MBOV07

04S00

004077

_at

7.23

0.0069

9Intergenic

region

367247–367

495

MBOV07

04S00

004078

_at

3.52

0.0364

Intergenic

region

370001–370

290

MBOV07

04S00

004100

_at

2.10

0.0353

Intergenic

region

450536–450

761

MBOV07

04S00

004129

_at

3.39

0.0454

Intergenic

region

572726–572

956

MBOV07

04S00

004140

_at

4.99

8.61E−0

5Intergenic

region

594243–594

378

MBOV07

04S00

004171

_at

3.36

0.0001

2Intergenic

region

695709–695

931

MBOV07

04S00

004172

_at

2.22

0.0007

24Intergenic

region

696274–696

740

MBOV07

04S00

004178

_at

2.08

0.0039

4Intergenic

region

709958–710

110

MBOV07

04S00

004205

_at

2.16

0.0071

6Intergenic

region

798730–799

092

MBOV07

04S00

004290

_at

2.73

0.0408

Intergenic

region

102675

3–10

2698

0

MBOV07

04S00

004310

_at

3.71

0.0085

8Intergenic

region

108800

8–10

8812

2

MBOV07

04S00

004322

_at

2.84

0.0016

6Intergenic

region

1120

652–1120

849

MBOV07

04S00

004372

_at

2.01

0.0006

53Intergenic

region

125327

1–12

5337

4

MBOV07

04S00

004388

_at

2.00

0.0073

6Intergenic

region

130042

5–13

0056

6

MBOV07

04S00

004412

_at

2.32

0.0022

3Intergenic

region

137488

9–13

7497

8

MBOV07

04S00

004418

_at

2.50

0.0029

8Intergenic

region

139308

2–13

9322

2

MBOV07

04S00

004440

_at

3.99

0.0059

6Intergenic

region

145253

9–14

5278

7

MBOV07

04S00

004446

_at

4.97

0.0283

Intergenic

region

147279

3–14

7290

5

MBOV07

04S00

004458

_at

2.10

0.0019

6Intergenic

region

150352

8–15

0364

7

MBOV07

04S00

004473

_at

5.70

0.0012

7Intergenic

region

156415

9–15

6429

6

MBOV07

04S00

004498

_at

5.40

0.0355

intergenic

region

1644

493–16

4459

8

MBOV07

04S00

004500

_at

3.03

0.0105

Intergenic

region

164750

1–16

4809

8

MBOV07

04S00

004502

_at

2.10

0.0461

Intergenic

region

165965

0–16

6005

0

MBOV07

04S00

004503

_at

20.89

0.0008

59Intergenic

region

166324

9–16

6339

9


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S00

004532

_at

2.08

0.0073

1Intergenic

region

176964

4–17

7001

2

MBOV07

04S00

004557

_at

2.47

0.0015

4intergenic

region

1867

500–18

6759

2

MBOV07

04S00

004558

_at

4.13

0.0421

Intergenic

region

187065

0–18

7084

2

MBOV07

04S00

004559

_at

2.23

6.92E−0

5Intergenic

region

187931

9–18

7942

6

MBOV07

04S00

004582

_at

2.02

0.0055

4intergenic

region

1966

948–19

6738

6

MBOV07

04S00

004606

_at

2.15

0.0055

3Intergenic

region

204035

3–20

4045

4

MBOV07

04S00

004632

_at

3.07

0.0003

99Intergenic

region

210400

2–21

0414

1

MBOV07

04S00

004662

_at

6.76

0.0086

2Intergenic

region

220896

3–22

0986

0

MBOV07

04S00

004668

_at

3.53

4.99E−0

5Intergenic

region

221861

3–22

1881

3

MBOV07

04S00

004683

_at

2.05

0.0191

Intergenic

region

226377

7–22

6389

1

MBOV07

04S00

004684

_at

5.00

0.0035

7Intergenic

region

226473

5–22

6494

5

MBOV07

04S00

004704

_at

2.53

0.031

Intergenic

region

236098

2–23

6110

7

MBOV07

04S00

004718

_at

4.80

0.0167

Intergenic

region

240315

2–24

0325

3

MBOV07

04S00

004719

_at

3.69

0.021

Intergenic

region

240472

7–24

0493

5

MBOV07

04S00

004752

_at

2.08

0.0006

54Intergenic

region

249519

4–24

9543

1

MBOV07

04S00

004755

_at

2.06

0.0079

3Intergenic

region

250843

5–25

0856

3

MBOV07

04S00

004768

_at

2.53

0.0070

4Intergenic

region

254212

7–25

4242

0

MBOV07

04S00

004784

_at

6.31

0.0002

85Intergenic

region

258055

7–25

8078

9

MBOV07

04S00

004800

_at

2.23

0.0363

Intergenic

region

264086

0–26

4095

7

MBOV07

04S00

004803

_at

5.81

0.0058

4intergenic

region

2648

165–26

4826

9

MBOV07

04S00

004836

_at

2.42

0.0061

5Intergenic

region

274977

6–27

5038

3

MBOV07

04S00

004862

_at

2.35

0.0269

Intergenic

region

283481

7–28

3522

6

MBOV07

04S00

004873

_at

2.21

0.0009

36Intergenic

region

286898

9–28

6926

7

MBOV07

04S00

004891

_at

2.57

7.12E−0

5Intergenic

region

291986

6–29

20211

MBOV07

04S00

004893

_at

2.31

0.0065

8Intergenic

region

292363

5–29

2377

8

MBOV07

04S00

004897

_at

2.10

0.0024

1Intergenic

region

292936

2–29

2953

5

MBOV07

04S00

004923

_at

2.21

0.0226

Intergenic

region

298522

3–29

8552

9

MBOV07

04S00

004942

_at

2.38

0.0209

Intergenic

region

3055611–30

55811

MBOV07

04S00

004993

_at

2.24

0.0304

Intergenic

region

326270

7–32

6282

8

MBOV07

04S00

004997

_at

4.35

0.0066

3Intergenic

region

326783

5–32

6793

4

MBOV07

04S00

005016

_at

2.55

0.0021

4Intergenic

region

331902

0 –33

1919

5

MBOV07

04S00

005021

_at

2.46

0.0053

2Intergenic

region

333284

7–33

3314

1

MBOV07

04S00

005049

_at

11.34

0.0068

3Intergenic

region

339346

0–33

9355

7

MBOV07

04S00

005051

_at

2.57

0.0058

5intergenic

region

341175

9–34

1269

6

MBOV07

04S00

005062

_at

2.76

0.0331

Intergenic

region

344077

5–34

4121

6

MBOV07

04S00

005064

_at

2.33

0.0146

Intergenic

region

344336

2–34

4351

7

MBOV07

04S00

005065

_at

2.74

0.0136

Intergenic

region

344589

2–34

4636

9

MBOV07

04S00

005081

_at

10.75

0.0258

Intergenic

region

350252

2–35

0280

8

MBOV07

04S00

005101

_at

7.30

0.0014

2Intergenic

region

354671

8–35

4694

9

MBOV07

04S00

005125

_at

5.82

0.0034

5Intergenic

region

364587

0–36

4610

1


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S00

005160

_at

2.38

0.0089

Intergenic

region

378767

1–37

8785

9

MBOV07

04S00

005172

_at

3.91

0.0052

5Intergenic

region

383171

6–38

3194

7

MBOV07

04S00

005207

_at

9.10

0.0003

77Intergenic

region

396433

8–39

6444

8

MBOV07

04S00

005211_at

2.01

0.0387

Intergenic

region

397652

4–39

7687

1

MBOV07

04S00

005223

_at

6.73

0.0118

Intergenic

region

402023

4–40

2033

6

MBOV07

04S00

005261

_at

2.18

0.0066

6Intergenic

region

4110

228–4110

318

MBOV07

04S00

005274

_at

3.07

0.0161

Intergenic

region

414618

3–41

4652

2

MBOV07

04S00

005281

_at

2.34

0.0496

Intergenic

region

416890

9–41

6912

5

MBOV07

04S00

005310

_at

3.28

0.0358

Intergenic

region

427200

8–42

7221

3

Region3:

genesdow

nregu

latedbyperacetic

acid

only:96

genes

AFFX-LysX-3_at

−2.53

0.0392

B.subtilis/GEN=lys/DB_X

REF=gb:X17013.1/

NOTE=SIF

correspondingto

nucleotid

es1061–

1343

ofgb

:X1701

3.1,

not10

0%identical/

DEF=B.subtilislysgene

fordiam

inop

imelate

decarbox

ylase(EC4.1.1.20

).AFFX-LysX-5_at

−2.39

0.0103


REF=gb:X17013.1/

NOTE=SIF

correspondingto

nucleotid

es387–

658of

gb:X17013.1,

not100%

identical/DEF=B.

subtilislysgene

fordiam

inop

imelate

decarbox

ylase(EC4.1.1.20

).AFFX-LysX-M

_at

−2.59

0.0079

5B.subtilis/GEN=lys/DB_X

REF=gb:X17013.1/

NOTE=SIF

correspondingto

nucleotid

es720–

990of

gb:X17013.1,

not100%

identical/DEF=B.

subtilislysgene

fordiam

inop

imelate

decarbox

ylase(EC4.1.1.20

).AFFX-PheX-5_at

−2.26

0.0349

B.subtilis/GEN=ph

eA/DB_X

REF=gb

:M24

537.1/

NOTE=SIF

correspondingto

nucleotid

es2028–

2375

ofgb

:M24

537.1,

not10

0%identical/

DEF=Bacillus

subtilissporulationprotein

(spoOB),GTP-binding

protein(obg

),phenylalaninebiosynthesisassociated

protein

(pheB),andmon

ofunctionalprephenate

dehydratase(pheA)genes,completecds.

AFFX-r2-Bs-thr-3_s_at

−2.60

0.0441

B.subtilis/GEN=thrB/DB_X

REF=gb:X04603.1/

NOTE=SIF

correspondingto

nucleotid

es1735–

2143

ofgb

:X0460

3.1/DEF=B.subtilisthrB

and

thrC

genesforho

moserinekinase

andthreon

ine

synthase

AFFX-r2-Bs-thr-M_s_at

−2.28

0.0319

B.subtilis/GEN=thrC,thrB/DB_X

REF=gb

:X04603.1/NOTE=SIF

correspondingto

nucleotid

es1045–155

6of

gb:X0460

3.1/DEF=B.

subtilisthrB

andthrC

genesforho

moserine

kinase

andthreon

inesynthase


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

AFFX-ThrX-3_at

−2.43

0.0243

B.subtilis/GEN=thrB/DB_X

REF=gb:X04603.1/

NOTE=SIF

correspondingto

nucleotid

es1689–

2151

ofgb

:X0460


and

thrC

genesforho

moserinekinase

andthreon

ine

synthase

(EC2.7.1.39

andEC4.2.99.2,

respectiv

ely).

AFFX-ThrX-5_at

−2.23

0.0332

B.subtilis/GEN=thrC/DB_X

REF=gb:X04603.1/

NOTE=SIF

correspondingto

nucleotid

es288–

932of

gb:X0460


and

thrC

genesforho

moserinekinase

andthreon

ine

synthase

(EC2.7.1.39

andEC4.2.99.2,

respectiv

ely).

MBOV07

04S00

000013

_s_at

BCG_0

013

−2.20

0.000339

Putativepara-aminobenzoatesynthase

component

IItrpG

trpG

_1Aminoacid

transport

andmetabolism

MBOV07

04S00

000040

_s_at

BCG_0

043

−2.28

0.0004

61Putativeanthranilate

synthase

compo

nent

IItrpG

trpG

_2Aminoacid

transport

andmetabolism

Coenzym

emetabolism

MBOV07

04S00

000074

_at

BCG_0

078c

−2.15

0.0176

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000092

_at

BCG_0

095A

−2.51

0.00329

Hypothetical

protein,

antitoxin

MBOV07

04S00

000164

_at

BCG_0

171c

−2.19

0.0001


protein

MBOV07

04S00

000170

_at

BCG_0

177c

−2.18

0.0005

06Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000183

_at

BCG_0

190c

−2.09

0.0016

2Putativeacyl-CoA

dehy

drog

enasefadE

2

MBOV07

04S00

000225

_at

BCG_0

233

−2.10

0.0003


protein

MBOV07

04S00

000289

_at

BCG_0

297c

−2.22

0.0068

8Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000290

_at

BCG_0

298c

−2.28

0.0055


protein

Transcriptio

nCoenzym

emetabolism

MBOV07

04S00

000291

_at

BCG_0

299c

−2.57

0.0189

Putativeintegralmem

branenitrite

extrusionprotein

narK

3Inorganiciontransport

andmetabolism

MBOV07

04S00

000371

_at

BCG_0

379

−2.11

0.0072

3Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000397

_at

BCG_0

405c

−2.16

0.0039

1Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000522

_at

BCG_0

530

−2.01

0.0008

73Putativeph

osph

oglycerate

mutase1gp

m1

gpm1

Carbohydratetransport

andmetabolism

MBOV07

04S00

000557

_at

BCG_0

565

−2.11

6.19E−0

5Putativegaba

perm

ease

gabP

(4-aminobutyrate

transportcarrier)

gabP

Aminoacid

transport

andmetabolism

MBOV07

04S00

000603

_at

BCG_0

613

−2.23

0.0030

2Putativecytochromep4

5013

5b1cyp1

35B1

cyp135

B1

Secon

dary

metabolites

biosynthesis,

transport,and

catabo

lism

MBOV07

04S00

000616

_at

BCG_0

626

−2.45

0.0017

7Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000622

_at

BCG_0

632

−2.24

0.000448

Hypothetical

integral

mem

braneproteinyrbE

2AyrbE

2ASecon

dary

metabolites

biosynthesis,

transport,and

catabo

lism

MBOV07

04S00

000685

_at

BCG_0

696c

−2.37

0.0496

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000777

_at

BCG_0

788

−2.07

0.0002

96Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000797

_at

BCG_0

808c

−2.46

0.0277

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

000853

_at

BCG_0

864

−2.18

0.00142

Putativeam

inoacid

aminotransferase

General

functio

npredictio

non

ly


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S0000

1050

_at

BCG_106

1c−2

.58

0.00

175

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1080

_at

BCG_109

1c−2

.20

0.0407

Two-component

transcriptionalregulatortrcR

trcR

Signaltransductio

nmechanism

sMBOV07

04S0000

1151

_at

BCG_116

3c−2

.11

0.00

168

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1152

_at

BCG_116

4−2

.17

0.000196

Putativepara-nitrobenzylesterase

Lipid

metabolism

MBOV07

04S0000

1158

_at

BCG_117

0−2

.41

0.0115

PutativelytB-related

proteinlytB2

lytB2

MBOV07

04S0000

1178

_at

BCG_119

0c−2

.27

0.0409

Putativetranscriptionalregulatorprotein

Transcriptio

nGeneral

functio

npredictio

non

ly

MBOV07

04S0000

1179

_at

BCG_119

1−4

.67

0.02

83Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1199

_at

BCG_121

2c−2

.29

0.0022


protein

Transcriptio

n

MBOV07

04S0000

1235

_at

BCG_124

9−2

.18

0.0392

Putativepyrroline-5-carboxylatedehydrogenase

rocA

rocA

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

1489

_at

BCG_150

3−2

.16

2.92

E−0

9Putativebiotin

sulfoxidereductasebisC

bisC

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

1507

_at

BCG_152

1−2

.04

0.000985


protein

Transcriptio

n

MBOV07

04S0000

1534

_at

BCG_154

8c−2

.19

0.00

776

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1580

_at

BCG_159

4c−2

.38

0.00

507

Putativehemog

lobinglbN

glbN

General

functio

npredictio

nonly

MBOV07

04S0000

1608

_at

BCG_162

2−2

.25

0.00

766

Putative8-am

ino-7-ox

onon

anoate

synthase

bioF

1bioF

1Coenzym

emetabolism

MBOV07

04S0000

1662

_at

BCG_167

6−2

.03

0.00

062

ExcinucleaseABC,subunitauv

rAuvrA

DNA

replication,

recombinatio

n,and

repair

MBOV07

04S0000

1671

_at

BCG_168

5−2

.07

0.01

74PEfamily

protein

MBOV07

04S0000

1823

_at

BCG_183

7c−2

.25

0.00

258

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1827

_at

BCG_184

1−2

.02

0.01

33PPEfamily

protein

MBOV07

04S0000

1866

_at

BCG_188

2c−2

.11

0.00717


protein

Transcriptio

n

MBOV07

04S0000

1874

_at

BCG_189

0c−2

.00

0.00

687

PutativeNADH

dehy

drog

enasend

hnd

hEnergyprod

uctio

nand

conv

ersion

MBOV07

04S0000

1880

_at

BCG_189

6−2

.20

0.000548

Alanine-andproline-rich

secreted

proteinapa

apa

MBOV07

04S0000

1883

_at

BCG_189

9c−2

.06

0.01

83Putativeintegral

mem

braneprotein

General

functio

npredictio

nonly

MBOV07

04S0000

1897

_at

BCG_191

3− 2

.02

0.00299

Putativeintegral

mem

braneprotein

Aminoacid

transport

andmetabolism

MBOV07

04S0000

1977

_at

BCG_199

4−2

.79

0.00

078

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1993

_at

BCG_200

9c−2

.19

0.00637

Putativemetal

catio


GctpG

ctpG


andmetabolism

MBOV07

04S0000

1994

_at

BCG_201

0c−2

.34

0.00

687

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1995

_at

BCG_2011c

−2.32

0.00646


protein

Transcriptio

n


()

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S0000

2071_at

BCG_208

8−2

.24

0.00

997

PutativeRNA

polymerasesigm

afactor,ECF

subfam

ily,SigC

sigC

Transcriptio

n

MBOV07

04S0000

2128_at

BCG_214

6c−2

.09

1.96

E−0

5Putativeoxidoreductase

Secondary

metabolites

biosyn

thesis,

transport,and

catabo

lism

MBOV07

04S0000

2253_at

BCG_227

1−3

.41

0.00463

Putativesecreted

unknow

nprotein

MBOV07

04S0000

2325_at

BCG_234

3c−2

.26

0.00771

Putativeornithineam

inotransferase

(N-terminus

part)rocD

1rocD

1Aminoacid

transport

andmetabolism

MBOV07

04S0000

2326_at

BCG_234

4c−2

.65

0.00

115

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2350_at

BCG_236

8−2

.11

0.00

0125

Putativetransm

embraneprotein

MBOV07

04S0000

2371_at

BCG_238

9−2

.18

0.01

93Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2384_at

BCG_240

2c−2

.12

0.00798

Putativeoxygen-independent

coproporphyrinogen

IIIox

idasehemN

hemN

Coenzym

emetabolism

MBOV07

04S0000

2399_at

BCG_241

7c−2

.07

0.00

174

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2502_at

BCG_252

3c−2

.06

0.00978

Putativesuccinyl-CoA

:3-K

ETOACID

-COENZYMEATRANSFERASEsubunitbeta

scoB

scoB

Lipid

metabolism

MBOV07

04S0000

2646_at

BCG_266

7c−2

.34

0.00158


protein

(probablyarsR

family

)Transcriptio

n

MBOV07

04S0000

2664_at

BCG_268

5−2

.11

0.00182

Putativesecreted

protease

General

functio

npredictio

nonly

MBOV07

04S0000

2668_at

BCG_268

9c−2

.00

0.00

443

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2674_at

BCG_269

5c−2

.25

0.00

156

Putative1-deoxy-

D-xylulose5-ph

osphatesynthase

dxs1

dxs1

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S0000

2705_at

BCG_272

6−2

.00

0.000285

Putativesolublepyridine

nucleotid

etranshydrogenase

sthA

sthA

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

2743_at

BCG_276

4c−2

.38

0.01

87Putativecelldivision

transm

embraneproteinftsK

ftsK

Celldivision

and

chromosom

epartitioning

MBOV07

04S0000

2773_at

BCG_279

4c−2

.04

0.00

317

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2797_at

BCG_281

8−2

.22

0.00

895

Putativehy

drolase

General

functio

npredictio

nonly

MBOV07

04S0000

2820_at

BCG_284

3c−2

.53

0.02

76Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

2980_at

BCG_300

3c−2

.04

0.0127

Putativeglycerol-3-phosphate

dehydrogenase

[NAD(P)+]gp

dA2

gpdA

2Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

3005_at

BCG_302

8−2

.06

0.00

203

Putativelip

oprotein

lppZ

lppZ

Carbohydratetransport

andmetabolism

MBOV07

04S0000

3047_at

BCG_307

0c−2

.20

0.00

0119

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3049_at

BCG_307

2c−2

.16

0.0315

Ribonucleoside-diphosphatereductasesubunitbeta

nrdF

2nrdF

2Nucleotidetransport

andmetabolism

MBOV07

04S0000

3084_at

BCG_310

7c−2

.54

0.0018

Virulence-regulatingtranscriptionalregulatorvirS

(araC/xylSfamily

)virS

Transcriptio

n

MBOV07

04S0000

3214_at

BCG_323

7c−2

.25

0.00

561

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3233_s_at

BCG_325

6c−2

.07

6.77

E−0

5Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3260_at

BCG_328

3−2

.22

0.01

38Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S0000

3275_at

BCG_329

8−2

.73

0.01

87Hyp

othetical

protein

Fun

ctionun

know

n


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

MBOV07

04S00

003276

_at

BCG_3

299

−2.19

0.00587

Putativemetal

catio

n-transportin

gP-typeatpase

CctpC

ctpC


andmetabolism

MBOV07

04S00

003398

_at

BCG_3

426

−2.16

0.0001

04Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

003573

_at

BCG_3

601

−2.01

0.0051

9Putativedehydrog

enase

Secon

dary

metabolites

biosynthesis,

transport,and

catabo

lism

MBOV07

04S00

003623

_at

BCG_3

651

−2.14

0.0073

9Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

003698

_at

BCG_3

726c

−2.42

0.0002

89Putativeprotease

MBOV07

04S00

003775

_at

BCG_3

803

−2.11

0.0020

1Transcriptio

nalregu

latory

protein(probablyarsR

family

)Transcriptio

n

MBOV07

04S00

003784

_at

BCG_3

812c

−2.15

0.0056

8Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

004157

_at

−2.14

0.0037

9Intergenic

region

643702–6

43835

MBOV07

04S00

004401

_at

−2.12

0.01

Intergenic

region

133256

1–13

3265

1

MBOV07

04S00

004664

_at

−2.06

0.0281

Intergenic

region

221229

3–22

1253

5

MBOV07

04S00

005076

_at

−2.72

0.0040

5Intergenic

region

349593

8–34

9607

3

Region4:

genesupregu

latedbybothsodium

hyp

ochlorite

andhyd

rogenperox

ide:

6genes

MBOV07

04S00

000169

_at

BCG_0

176

9.37

0.0002

85Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

002466

_at

BCG_2

486c

13.11

0.00113

Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

003056

_at

BCG_3

079c

7.26

0.0043

5Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

003500

_at

BCG_3

528

9.92

0.0002

62Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

003874

_at

BCG_3

902

3.69

0.0005

11Hyp

othetical

protein

Fun

ctionun

know

n

MBOV07

04S00

004610

_at

3.26

7.56E−0

5Intergenic

region

204784

5–20

4802

4

Region5:

genesupregu

latedbybothsodium

hyp

ochlorite

andperacetic

acid:20

genes

AFFX-PheX-M

_at

2.30

0.0053

8B.subtilis/GEN=ph

eA/DB_X

REF=gb

:M24

537.1/

NOTE=SIF

correspondingto

nucleotid

es2437–

2828

ofgb

:M24

537.1/DEF=Bacillus

subtilis

sporulationprotein(spoOB),GTP-binding

protein(obg),phenylalaninebiosynthesis

associated

protein(pheB),andmon

ofunctional

prephenate

dehydratase(pheA)genes,complete

cds.

AFFX-r2-Bs-lys-5_at

2.44

0.00666


REF=gb:X17013.1/

NOTE=SIF

correspondingto

nucleotid

es411–

659of

gb:X17013.1,

not100%

identical/DEF=B.

subtilislysgene

fordiam

inop

imelate

decarboxylase(EC4.1.1.20

).AFFX-r2-Bs-thr-5_s_at

2.04

0.025

B.subtilis/GEN=thrC/DB_X

REF=gb:X04603.1/

NOTE=SIF

correspondingto

nucleotid

es290–

888of

gb:X0460


and

thrC

genesforho

moserinekinase

andthreon

ine


Tab

le3

(con

tinued)

Affym

etrixprobeID

ORFno.

10min

Descriptio

nSym

bol

Functionalclass

Foldchange

Pvalue

synthase.

AFFX-ThrX-M

_at

2.20

0.016

B.subtilis/GEN=thrC,thrB/DB_X

REF=gb

:X04603.1/NOTE=SIF

correspondingto

nucleotid

es99

5–15

62of

gb:X0460

3.1,

not10

0%identical/DEF=B.subtilisthrB

andthrC

genesfor

homoserinekinase

andthreon

inesynthase

(EC

2.7.1.39

andEC4.2.99.2,respectiv

ely).

MBOV07

04S0000

0607

_at

BCG_061

7c2.35

0.0003

28Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

1758

_at

BCG_177

2c2.62

0.0001

64Putativetransm

embraneprotein

MBOV07

04S0000

1998

_at

BCG_201

42.14

2.65E−0

5Putativemetal

catio


ActpF

ctpF


andmetabolism

MBOV07

04S0000

2033

_at

BCG_204

9c2.08

0.0022

2Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

2035

_at

BCG_205

12.47

0.0002

31Hyp

othetical

proteinAcg

Energyprod

uctio

nand

conv

ersion

MBOV07

04S0000

2631

_at

BCG_265

2c2.48

0.0037

Putativetransm

embranealanine-

andleucine-rich

protein

General

functio

npredictio

nonly

MBOV07

04S0000

2632

_at

BCG_265

3c3.38

7.35E−0

5Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

2633

_at

BCG_265

4c2.38

0.0004

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

2634

_at

BCG_265

52.24

2.23E−0

5Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

3020

_at

BCG_304

3c2.02

0.0099

7PEfamily

protein

MBOV07

04S0000

3055

_at

BCG_307

8c3.19

0.0018

9Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

3126

_at

BCG_314

92.61

0.0149

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

3130

_at

BCG_315

3c2.31

0.0005

84Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

3131

_at

BCG_315

42.30

0.0014

Hyp

othetical

protein

Functionun

know

n

MBOV07

04S0000

3176

_at

BCG_319

92.97

0.0038

7Putativeam

idase

Translatio

n,ribosomal

structure,

and

biog

enesis

MBOV07

04S0000

4478

_at

2.06

0.0074

4Intergenic

region

1572

691–15

7299

1

Region5:

genes

dow

nregu

latedbybothsodium

hyp

ochlorite

andperacetic

acid:1gene

MBOV07

04S0000

1742

_at

BCG_175

6−2

.24

0.0072

3Hyp

othetical

protein

Functionun

know

n

Region6:

genes

upregu

latedbybothhyd

rogenperox

idean

dperacetic

acid:3genes

MBOV07

04S0000

1931

_at

BCG_194

7c5.52

1.29E−0

5Catalase-peroxidase-peroxynitritase

TkatG

katG


andmetabolism

MBOV07

04S0000

2377

_at

BCG_239

5c3.22

0.0027

9Polyk

etidesynthetase

mbtD

mbtD

Metabolites

biosynthesis,

transport,and

catabolism

MBOV0704S00002382_at

BCG_2400c

2.11

0.00387

Putativeisochorism

atesynthase

mbtI

mbtI

Aminoacid

transport

andmetabolism

Coenzym

emetabolism

The

microarrayresults

arethemeanof

threereplicates

ofeach

gene.The

fold

change

isapo

sitiv

enu


levelin

theexperimentincreasedcomparedto

thecontrolandisa

negativ

enu


levelin

theexperimentdecreasedcomparedto

thecontrol


uvrA and nrdF2 are directly regulated by the RecA-NDppromoter. In contrast to these results, uvrA has been shownto be induced in M. tuberculosis treated with mitomycinwhich is a DNA damaging agent (Brooks et al. 2001).

The discussion from this point on focuses on the comparisonof the M. bovis BCG response to the oxidative disinfectants,peracetic acid, hydrogen peroxide, and sodium hypochloriteand provides conclusions to the two sections of the study.

Discussion: comparisons among the toxicogenomicresponses of M. bovis BCG to sodium hypochlorite,hydrogen peroxide, and peracetic acid

Table 3 contains the genes found in the seven regions of theVenn diagram (Fig. 4). However, in this discussion, wehave focused on the genes in the intersecting regionsamong the three disinfectants (regions 4, 5, and 6) since ourprior publications (Jang et al. 2009a, b) and part of thecurrent study have described in detail the toxicogenomicresponses of M. bovis BCG to sodium hypochlorite,hydrogen peroxide, and peracetic acid.

Region 4: genes up- and downregulated in commonbetween sodium hypochlorite and hydrogen peroxide

Five of the upregulated genes in this region were hypotheticalproteins and the sixth gene was an intergenic region with noknown function.

Region 5: genes up- and downregulated in commonbetween sodium hypochlorite and peracetic acid

The ctpF gene which encodes a putative metal cationtransporter P-type atpase A was the only gene with a knownfunction in this region. Interestingly, in another study, thectpF gene was upregulated in M. tuberculosis in response toexposure to reactive nitrogen intermediates (Ohno et al.2003). Further, the ctpF gene was upregulated in M.tuberculosis in response to growth in a hypoxic environment(Sherman et al. 2001).

Region 6: genes up- and downregulated in commonbetween peracetic acid and hydrogen peroxide

The three upregulated genes in this region were the katGgene which encodes a catalase-peroxidase-peroxynitritase Tenzyme and the mbtD and mbtI genes which encodepolyketide synthases involved in the biosynthesis of myco-bactins. As earlier mentioned, katG is an anti-oxidative stressenzyme produced in mycobacteria to counteract the effectsof reactive oxygen intermediates. Mycobactins are salicylicacid-derived siderophores, important in mycobacterialiron acquisition/virulence. These results further empha-

size the intricate connection between iron regulation andoxidative stress response in M. bovis BCG exposed toboth disinfectants.

Region 7: no genes were upregulated in commonbetween sodium hypochlorite, hydrogen peroxide,and peracetic acid

In conclusion, the first section of this report suggests that theregulation of arginine levels and virulence factors may play anadaptive role against peracetic acid treatment in M. bovisBCG. The results from this section also suggest that, inaddition to the upregulation of katG which plays a major rolein defense against oxidative damage, cell wall modificationdue to upregulation of genes coding for cell wall compo-nents after 20 min may also function as a protective strategyagainst peracetic acid damage. The downregulation of DNArepair genes after both 10 and 20 min indicates that theinhibition of DNA repair may contribute to the mechanismof action of peracetic acid. Further, the upregulation of thedevR–devS signal transduction system, which is a regulatorof the genetic response of M. tuberculosis in oxygen-deficient environments after 10 min, indicates that thissystem plays a role in the early adaptive response of M.bovis BCG to peracetic acid-induced oxidative stress. Insummary, the complex interplay of the changes in thesemetabolic processes may contribute to both the inhibitoryeffect of peracetic acid on M. bovis BCG and the resistancestrategies utilized by M. bovis BCG against the effect ofperacetic acid treatment. This is the first report of thegenome-wide response of M. bovis BCG to peracetic acidtreatment and therefore advances the understanding of themode of peracetic acid in M. bovis BCG. The second sectionof this study reports that iron acquisition/virulence is affectedin M. bovis BCG in response to hydrogen peroxide andperacetic acid treatment. This comparative analysis alsodetermined that the ctpF gene was upregulated in response toboth peracetic acid and sodium hypochlorite treatment. Thiscomparative analysis helps in the identification of commonlyactivated genes between these oxidative antimicrobials,which further improves the understanding of their modesof action in mycobacteria. The information generated in thisstudy will benefit other researchers studying the transcrip-tomic response of mycobacteria to peracetic acid specificallyand to oxidative biocides in general.

Acknowledgment This research is supported by the United StatesEnvironmental Protection Agency Grant number T-83284001-4.Although the research described in this paper has been funded

wholly by the United States Environmental Protection Agency, it hasnot been subjected to the Agency’s peer and administrative review andtherefore may not necessarily reflect the views of the EPA nor doesthe mention of trade names or commercial products constituteendorsement of recommendation of use.


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