Cleeton, Rebecca

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Digital Kenyon: Research, Scholarship, and Creative ExchangeKenyon Summer Science Scholars Program Summer Student Research ScholarshipSummer 2008Permeant Acid-Dependent Gene Expression inBacillus subtilisRebecca CleetonFollow this and additional works at: https://digital.kenyon.edu/summerscienceprogramPart of the Mathematics CommonsThis Poster is brought to you for free and open access by the Summer Student Research Scholarship at Digital Kenyon: Research, Scholarship, andCreative Exchange. It has been accepted for inclusion in Kenyon Summer Science Scholars Program by an authorized administrator of Digital Kenyon:Research, Scholarship, and Creative Exchange. For more information, please contact noltj@kenyon.edu.Recommended CitationCleeton, Rebecca, "Permeant Acid-Dependent Gene Expression in Bacillus subtilis" (2008). Kenyon Summer Science Scholars Program.Paper 425.https://digital.kenyon.edu/summerscienceprogram/425Permeant Acid-Dependent Gene Expression in Bacillus subtilisRebecca Cleeton ’09, Jessie Wilks ’08, Ryan Kitko, Brian D. Jones, Joan L. SlonczewskiKenyon College Department of BiologyGene expression of Bacillus subtilis grown under conditions of 0 mM and 30 mMbenzoate was measured using microarray hybridization. An increase in theconcentration of benzoate in B. subtilis cultures is a means of lowering theinternal pH of the organism. The concentrations of benzoate used weredetermined by a growth curve testing 0, 10, 30, and 60 mM concentrations ofbenzoate at external pH 7.0. Four biological replicates from 0 mM and 30 mMconcentrations were used for the 5006 gene microarray hybridization, performedat the University of Wisconsin-Madison’s Gene Expression Center. Real-timePCR was conducted to verify a few of the genes showing the largest expressionratios and a preliminary analysis of the data obtained from the microarray isunderway. Current analyses of these data suggest that 92.6% of the genes thatare highly expressed in response to an increase in internal pH are not similarthose that were found to be highly expressed as a response to increases in theexternal pH. These preliminary results are consistent with the original hypothesisthat a decrease in the internal pH of B. subtilis causes differential geneexpression.AbstractIntroductionMethodsResults & DiscussionReferencesAcknowledgements1. Azarkina, N. and Konstantinov, A. 2002. Stimulation of menaquinone-dependant electrontransfer in the respiratory chain of Bacillus subtilis by membrane energization. Journal ofBacteriology, 184: 5339 – 5347.2. Kosono, S., Morotomi, S., Kitada, M., and Kudo, T. 1999. Analyses of a Bacillus subilishomologue of the Na+/H+ antiporter gene which is important for pH homeostasis ofalkaliphilic Bacillus sp. C-125. Biochimica et Biophysica Acta, 1409: 171 – 175.3. Kovacs, T., Hargitai, A., Kovacs, K., and Mecs, I. 1998. pH-dependent activation of thealternative transcriptional factor sB in Bacillus subtilis. FEMS Microbiology Letters, 165: 323– 328.4. Krulwich, T., Quirk, P., and Guffanti, A. 1990. Uncoupler-reistant mutants of bacteria.Microbiological Reviews, 54: 52 – 65.5. Thomassin, S., Jobin, M., and Schmitt, P. 2006. The acid tolerance response of Bacilluscereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH. Archivesof Microbiology, 186: 229 – 239.6. Wilks, J., Kitko, R., Cleeton, S., Lee, G., Ugwu, C., Jones, B., BonDurant, S., andSlonczewski, J. Submitted to Applied Environmental Microbiology.I would like to thank Professor Joan L. Slonczewski for her guidance and training concerningmy project. I would also like to thank Jessie Wilks ‘08 and Ryan Kitko for their assistance andpatience every step of the way. Lastly, I would like to thank Professor Brian D. Jones for hishelp with the statistical analysis of the microarray data and the Gene Expression Center at theUniversity of Wisconsin-Madison for running my microarray chip.RNA was extracted using a Qiagen RNeasy kit and checked with a NanoDropspectrophotometer. RNA samples were stored at - 80 C before being sent to theGene Expression Center at the University of Wisconsin-Madison.cDNA was synthesized from the extracted mRNA with reverse transcriptaseusing standard procedures for Affymetrix GeneChips. cDNA was thenfragmented, N-terminusly labeled with Biotin, and hybridized to the microarraychip using standard protocols. Chips were scanned and then analyzed using thestatistical software D-chip.Real-time PCR was conducted using standard procedures for 25 µl samples in96-well plates with SYBR Green detector. Initial temperature of the thermocyclerwas 48.0 degrees C and the initial trial was run for 30 minutes. Samples wereanalyzed using 7500 software.pH, the measure of basicity and acidity, is internally regulated in mostorganisms. The maintenance of internal pH involves the upregulation andrepression of a number of genes.Bacillus subtilis is an excellent model organism for studying gram-positivebacteria, such as Bacillus anthracis (cause of anthrax). This organism is relativelylarge and undergoes a sporulation cycle, allowing for the observation of pHthroughout different parts of development.Microarray studies conducted last year by Wilks et al. indicated certain genesthat were upregulated under external acidic conditions. Questions were posed asto what genes are upregulated under internal acidic conditions.Results & Discussion Cont.MethodsGrowth curve experiments were conducted on B. subtilis using 2M benzoate,2M sodium acetate, and 2M methylamine hydrochloride. From these curves itwas determined that an RNA extraction would be conducted using 0 mM and 30mM concentrations of benzoate from a sodium salt of benzoate for real-time PCRand microarray experiments.A benzoate RNA extraction was completed at pH 7.0. Microarrays were sent tothe Gene Expression Center at the University of Wisconsin-Madison.B. subtilis strain MMB8 were grown in an LBK medium containing 50 mM MOPSbuffer and the external pH was adjusted to 7.0 with the addition of 5M KOH.Overnight cultures grew for 18 hours before they were diluted 1:500 for the waterbath. There were 4 biological replicates for each benzoate concentration tested(0 mM and 30 mM).Figure 1. B. subtilis growth over time in response to varying concentrations ofbenzoate.Growth curve experiments using benzoate, sodium acetate, and methylaminehydrochloride allowed for the determination of acid to use as well as possibleconcentrations. 0 mM and 30 mM were chosen for the microarray study.Real-time PCR was conducted on the following genes: trkA, katA, alsS, alsD,cadA, yrpC, maeN, yvgW, yqjM, and sigB. These genes were selected becausethey were upregulated in the presence of external HCl, based on the results oflast year’s microarray. These real-time PCR’s were not found to be significantlyupregulated under higher concentrations of benzoate.There were 526 genes significant from a 5006 gene microarray.The microarray study comparing 0 mM and 30 mM concentrations off benzoate showed only a 7.046% gene overlap with the microarray study conducted on external acidic conditions using HCl.Real-time PCR was also started on padC, citZ, acsA, and feuC based on the findings from my own microarray examining internal acidic conditions.Table 1. Most highly induced genes in B. subtilis in thepresence of 30 mM benzoate.padC: encodes aproduct (phenolicacid decarboxylase)that breaks downcoumerate, an acidproduced by plantsthat is found in thesoil and isstructurally similarto benzoate.ascA: codes for acetyl-coA synthetase. This could help remove more acetic acid,it may be that CoA can be used in metabolism without requiring the proton motiveforce.feuC: codes for an iron-uptake system. In the HCl microarray the acid made theplasma membrane more permeable to metals. The results was that a number ofmetal pumps were highly expressed in response to the influx of toxic metals. Inthe case of benzoate, however, it is possible that feuC is upregulated becausemore heme rings are needed in he cytoplasm for electron transport for increasedATP production.citZ: encodes citrate synthase II (major subunit). This enzyme uses acetic acidand oxaloacetic acid to create citrate, an intermediate within the Krebs cycle.benzoatecoumaric acid0.0010.010.111060 110 160 210 260log ODTime (min)Benzoate Growth Curve, pH 7.00 mM (1)0 mM (2)0 mM (3)10 mM (1)10 mM (2)10 mM (3)30 mM (1)30 mM (2)30 mM (3)60 mM (1)60 mM (2)60 mM (3)GeneMean Log2 RatioFunctionpadC -4.777 Phenolic acid decarboxylaseoppB -3.872 Oligopeptide ABC transporter, initiation of ilvN -3.76 Acetolactate synthesis (small subunit) citZ -2.63 Citrate synthase II (major)mpr -2.41 Extracellular metalloproteaseacsA -2.225 Acetyl-coA synthetasepurF -2.182 Purine biosynthesisald -2.105 L-alanine dehydrogenaseleuA -2.077 Leucine biosynthesisamhX -1.868 AmidohydrolasefeuC -1.678 Iron-uptake systemsucD -1.493 Succinyl-coA synthetase (alpha subunit)serC -1.493 Serine biosynthesis

Permeant Acid-Dependent Gene Expression in Bacillus subtilis

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Permeant Acid-Dependent Gene Expression in Bacillus subtilis

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