Research Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis

http://genomebiology.com/2001/2/6/research/0019.1 Research Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis )C EAI =5A MI = 5J FD= A4 >E V A= =?GKAI,=K@E V ) =E 0 =KJ W = @) J E A,=?DE )@@HAIIAI 0 C C7 ELAHIEJO2=IJAKH4AIA=H?D+A JAH 2 BK = 0 C C V 1 IJEJKJ =JE = )CH EGKA2=HEI /HEC %#! 2=HEI+A@AN# H=?A W /A A= @1 B H =JE?I 7 ELAHIEJ @A8AHI=E AI 5=E J 3KA JE %&!#8AHI=E AI+A@AN H=?A /A AJE?I B *=?JAHE= /A AI 1 IJEJKJ2=IJAKH %#% "2=HEI+A@AN# H=?A + HHAIF @A?A ) J E A,=?DE - =E =@=?DE (D K?? D K D =@=?DE (F=IJAKH BH Published: 1 June 2001 Genome Biology 2001, 2(6):research0019.1 0019.12 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2001/2/6/research/0019 2001 Sekowska et al., licensee BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914) Abstract Background: In global gene expression profiling experiments, variation in the expression of genes of interest can often be hidden by general noise. To determine how biologically significant variation can be distinguished under such conditions we have analyzed the differences in gene expression when Bacillus subtilis is grown either on methionine or on methylthioribose as sulfur source. Results: An unexpected link between arginine metabolism and sulfur metabolism was discovered, enabling us to identify a high-affinity arginine transport system encoded by the yqixyz genes. In addition, we tentatively identified a methionine/methionine sulfoxide transport system which is encoded by the operon ytmijklmhisp and is presumably used in the degradation of methionine sulfoxide to methane sulfonate for sulfur recycling. Experimental parameters resulting in systematic biases in gene expression were also uncovered. In particular, we found that the late competence operons come, comf and comg were associated with subtle variations in growth conditions. Conclusions: Using variance analysis it is possible to distinguish between systematic biases and relevant gene-expression variation in transcriptome experiments. Co-variation of metabolic gene expression pathways was thus uncovered linking nitrogen and sulfur metabolism in B. subtilis. Background /A AFH CH= ID=LAK? LAHA@=L=IJ K >AH BCA AI B K M BK?JE FH FJE C AMJOFAI BBK?JE = E LAI JEC=JE 1? K?JE MEJD ANFHAIIE FH BE E C ANFAHE A JI M A@CA B CA A IAGKA?AI FH LE@AI = ANJHA A O F MAHBK =FFH =?D J E JAHFHAJE C C >= CA A ANFHAIIE F=JJAH I 2=H=@ NE?= OD MALAH E JAHFHAJE C= JDAE B H =JE F JA JE= O=L=E => ABH JDAIAANFAHE A JI Received: 26 March 2001 Revised: 4 May 2001 Accepted: 4 May 2001 EI DE @AHA@ >O JDA LAHO =??KH=?O B JDA FDOIE?= A=IKHA A JI E L LA@ I JD=J JDA E F HJ=?A B JDA B K?JK=JE I E DAHA JJ >E CE?= ANFAHE A JIEI BJA @EIHAC=H@A@ 6DEI =?? K JIB HJDAA FD=IEIF =?A@>OI A IE F A@AI?HEF JELAIJK@EAI BCA AANFHAIIE MDAHAF=JJAH IIFA?EBE?J = F=HJE?K =HIAJ BCA AJE? HA LEH A J=? @EJE I=HA@EI F =OA@ KIK= O B H @E=C IJE? FKHF IAI HJK =JA O JH= I?HEFJE FH BE E C?= JA KI K?D HA = @?= DA F J comment reviews reports deposited research refereed research interactions information

2 Genome Biology Vol 2 No 6 5A MI =AJ= K? LAH AMCA ABK?JE I *AB HA K FE CJ?? KIE I D MALAH EJEI A?AII=HO MDA >IAHLE CL=HE=JE E = =HCA K >AH BCA AI J >A=> AJ @EIJE CKEIDIFA?EBE?>E CE?= ABBA?JIBH >=? CH K @ EIA= @IOIJA =JE?L=HE=JE I@KA J JDAFH?A@KHAEJIA B 1 JDAFHAIA JM H MDE?DKIAI*=?E KIIK>JE EI =I= @A MAD=LA= = O A@EJIJH= I?HEFJ AANFHAIIE E? @EJE I?D IA J OEA @ ANJHA A O IE E =H JH= I?HEFJE F=JJAH I CH MJDE JDAFHAIA?A BAEJDAH AJDE E A H AJDO JDE HE > IA 64 =I JDA IK BKH I KH?A 9A @EI? LAHA@ JD=J ALA MEJD = JDA >=? CH K @? IJH=E JI =?JE C JDA ANFAHE A JI= @JDAIE E =HEJOE JDACH MJD? @EJE I MA? K @ ANJH=?J >E CE?= O IEC EBE?= J >IAHL=JE I = A O JD=J JDAHA ANEIJA@ = IJH C E >AJMAA IK BKH AJ=> EI = @=HCE E A AJ=> EI 9A= I ID MJD=J E =@@EJE J BE @E C >E CE?= O HA AL= J F=JJAH I JDA E L K J=HO JHEC CAHE C B JH= IEJE I E A LEH A J=? @EJE I MEJD JDA HACK =JE?=I?=@AIJDAOE @K?AF =OI= E F HJ= JH AE?HA=JE C IOIJA =JE? >E=IAI E JDA ANFHAIIE B I A FAH I 6DA? F AN>E?DA EIJHO B?, )IO JDAIEIBH 4 )EI= I =IEC EBE?= JI KH?A BIOIJA =JE?AHH HI MDE?D = AI EJ LAHO @EBBE?K J J? F=HA ANFAHE A JI FAHB H A@ K @AH@EBBAHA J? @EJE I ALAHJDA AII MAMAHA=> AJ IAJKF=IE F A= @H >KIJIJ=JEIJE?= FH J? B HJH= I?HEF J A @=J= = = OIEI JD=J A => AI A J @EIJE CKEID B K?JK= JE IE ANFHAIIE BJDACA AI BE JAHAIJBH B K?JK=JE I @KAJ F=H=IEJE?ABBA?JI 6DEIFH J? @ AI JHAGKEHA>=? CH K @ EIA IK>JH=?JE H H = E =JE B JDA @=J= = J HE KI O@EBBE?K J= @? JH LAHIE= J=I! 9AD FAJD=J E =@@EJE J FH LE@E CK ANFA?JA@E B H = JE => KJ IK BKH AJ=> EI E *=?E KI IK>JE EI = FHALE KI O K HA? H@A@ HA =JE IDEF >AJMAA JDA AJ=> EI B =HCE E A = @ JD=J B AJDE E A =I MA =I AJ=> EI B AJDE E A IK B NE@A JDEI M H ME @H=M JDA =JJA JE B JDA A?K =H >E CO? K EJO J JDA HEI I B E? HHA?J E JAHFHAJ=JE E DAHA J E JDA =HCA >E CE?= L=HE=>E EJO B JH= I?HEFJ AANFAHE A JI Results Effect of the sulfur source on gene expression 8=HE=?A= = OIEIID MA@JD=JJDA IJE F HJ= JI KH?A B >E=IE? F=HE C@EBBAHA JANFAHE A JIEI=@EBBAHA?AE JDA = K J B4 )KIA@J IO JDAIE A?, ) E IA? @F =?AEI JDA@EBBE?K JO BAN=?J OHAFH @K?E CANFAHE A J=? @EJE I BH A ANFAHE A J J = JDAH IAA =JAHE= I = @ AJD @I )I ANFA?JA@ L=HE=?A = = OIEI ID MA@ JD=J?D= CE C JDA IK BKH I KH?A BH AJDE E A J 64 D=I EJJ AABBA?J JDAJH= I?HEFJE ALA B IJCA AI 1 JAHAIJ E C O D MALAH JDA EIJ B JDA IJ L=HE=> A CA AI D=I IALAH= @EIJE?JELA BA=JKHAI 1 JDEI CH KF CA AI B M BK?JE =HA IEC EBE?= J O HA K AH KI JD= ANFA?JA@ BH JDAEHFH F HJE E JDACA A MEJDF $ KHJDAH HAJDAIACA AI? HHAIF @J? HHA =JA@BK?JE I /H MJD E AJDE E A EI?D=H=?JAHE A@ > JD >O JDA DECDAH ANFHAIIE BJDA=HCE E A AJ=> E?F=JDM=O= @>OJD=J B FAH OGE:; MDE?DEIJD KCDJJ? @AB H= = E =?E@ )*+ FAH A=IA BK?JE MDAHA=I CH MJD 64 =?JEL=JAI IALAH= CA AI E L LA@ E = ALE=JE C NOCA J NE?EJO?=J= =IA DO@H FAH NE@AHA@K?J=IA NOCA =IA =IMA =I= C FAH A? @E C = FAH A=IA = @ M J >A? A?JA@ MEJD IK BKH AJ=> EI OJ 1 DEI2OJ OJ 1 HE>4DEF OJ " # 6DKI IALAH= CA AICH KFJ CAJDAHE = FDOIE CE?= O? IEIJA J FE?JKHA NE@ HA@K?JE =HCE E A>E IO JDAIEI )*+FAH A=IAI = @I 4A =H => O CA AIFKJ=JELA OANFHAIIA@=I= FAH $ =FFA=HJ CAJDAH E JDA EIJ OGE:; =HC/0OJ, =HC, OJ 1 2OJ E = O IALAH= CA AI>A CJ JDAFHA?A@E C EIJ= @=HA= I A >AHI B = FKJ=JELA FAH? /+? /, OG - 6=> A IK =HE AIJDAE B H =JE =L=E => A JDAIACA AI Arginine biosynthesis 1? JH=IJJ JDAIEJK=JE E = O JDAH>=?JAHE= JDA=HCE E A>E IO JDAIEICA AIE * IK>JE EI=HA IF EJE J JM F=HJI =HC- JD KCDJ J? @A B H =?AJO H EJDE A @A=?AJO =IA -+! # $ EIF=HJ B=JDEH@ FAH AII? A=H O E A@J =HCE E A AJ=> EI 6DEI IKCCAIJI I A? F=HJ A J= E =JE B JDA? HHAIF @E C AJ=> EJAI =I E AK =HO JAI MDAHA =HCE E A >E IO JDAIEI EI IF EJ >AJMAA JDA EJ?D @HE= = @ JDA?OJ F =I 8=HE=?A = = OIEI ID MI JD=J > JD IAJI BCA AI=HAIJE K =JA@MDA?A ICH M AJDE E A =IJDAIK BKHI KH?A6=> A 7IE CJDA=>I KJAL= KAIB HDO>HE@E =JE E JDAHAIFA?JELA? @EJE I MA B K @ JD=J JDA F=JDM=O E? K@E C?EJHK E A H EJDE A=?AJO JH= IBAH=IA-+!!# =?AJO H EJDE A = E JH= IBAH=IA -+ $ = @ H EJDE A?=H >= O JH= IBAH=IA-+!!M=IIJE K =JA@ $ B @ = @ JDA F=JDM=O A=@E C BH?EJHK E A = @ E? K@E C =HCE E A =HCE E IK??E =JA IO JD=IA -+ $! " # =HCE E IK??E =JA O=IA -+ "! = @ ;J, MDE?D D=I = K M HACK =J HO BK?JE M=I IJE K =JA@ % B @ 6=> A ;J,EI=I = DO@H FDE E?FH JAE HA =H => AB H EJI DECD? JA J B AJDE E A = @ EI JDAHAB HA K KIK= O HE?DE IK BKH 6DA FAH =HC/0OJ, EIB K @=I=MD AE JDA EIJ E 6=> A MDAHA=I B H JDA FAH =HC+ *,?=H)*=HC A BE @I O CA AI =HC =HC, = @ =HC E 6=> A 6DEIFH FJA@KIJ =JJDA=>I KJAL= KAIB K @ B H= CA AI BJDA FAH? IEIJA JMEJD LAHANFHAIIE B JDA FAH MA B K @ JD=J = JDAH CA AI E JDEI FAH AN?AFJ?=H) MAHA = I LAHANFHAIIA@ E AJDE E A 6=> A 6DAO @ J ID M KF = C JDA BEHIJ LAHAN FHAIIA@CA AIIE F O ME CJ JDAB=?JJD=JJDAHAM=I HA L=HE=>E EJO BH ANFAHE A J J ANFAHE A J B H JDAIA CA AI HAIK JE C E = MAH F IEJE E JDA L=HE=?A = = OIEI EIJ 6= A J CAJDAH D MALAH JDAIA HAIK JI ID M JD=J > JD FAH I=HAE @AA@ LAHANFHAIIA@ =I=MD A E AJDE E A CH MJD? @EJE I )I=? JH MA >IAHLA@JD=JJDAHAM=I @EBBAHA?AE ANFHAIIE BJDA=HC5 CA A MDE?DEIFHAIA J E = A JEHA O@EBBAHA J FAH BK M BK?JE

http://genomebiology.com/2001/2/6/research/0019.3 Table 1 Function of genes showing the largest differences as a function of the sulfur source Gene Mean Standard Function deviation argd 2.378 1.032 Arginine biosynthesis argf 2.874 0.735 Arginine biosynthesis argg 1.911 0.411 Arginine biosynthesis argh 3.414 1.139 Arginine biosynthesis argj 3.252 0.873 Arginine biosynthesis comgc 1.841 0.730 Late competence operon required for comgd 2.149 0.628 Late competence operon required for levd 2.653 0.757 PTS system - fructose-specific IIA component ycbd 2.071 0.452 Probable aldehyde dehydrogenase yqix 3.052 0.297 Probable amino-acid ABC permease (binding protein) yqiy 2.351 0.744 Probable amino-acid ABC permease (permease) yqiz 3.178 0.659 Probable amino-acid ABC permease (ATP-binding protein) yqze 2.000 0.946 Hypothetical protein ytzd 3.378 0.839 Hypothetical protein, downstream gene of operon arggh ahpf -3.013 0.601 Alkyl hydroperoxide reductase (large subunit) - reduces organic hydroperoxides in their reduced dithiol form kata -1.964 0.914 Vegetative catalase ytmi -2.418 0.489 Possible methionine sulfoxide acetyl transferase (operon ytmijklmhispytmoytnijribrhipoytnm) ytmj -2.480 0.787 Methionine - methionine sulfoxide ABC permease (binding protein) (this paper) ytml -1.852 0.979 Methionine - methionine sulfoxide ABC permease (permease) (this paper) ytmo -2.258 0.439 Methionine sulfoxide monooxygenase (this paper) The yqixyz permease 1J M=I ANFA?JA@ JD=J FAH A=>E EJO J AJDE E A M K @ >A =BBA?JA@E KJ= JI BI A BJDACA AIE L LA@E CH MJD AJDE E A )I JDA OGE:; CA AI D=LA IEC EBE?= J IE E =HEJOJ = E =?E@FAH A=IACA AI MAE LAIJEC=JA@FAH A =>E EJO B KJ= J >=?JAHE= J AJDE E A = @ =HCE E A 9A @E@ J BE @ = O @EBBAHA?A E AJDE E A =L=E =>E EJO E KJ= JI MEJD @EIHKFJE I E JDA FKJ=JELA FAH A=IA CA AI OJ DEI2 HOGE:; @=J= JID M 1? JH=IJ MA B K @ JD=J CH MJD B KJ= JI E = O B JDA OGE:; CA AI M=IIJH C OE F=EHA@E M??A JH=JE I B=HCE E A=I= I A EJH CA I KH?A 6DA >IAHL=JE JD=JCH MJD AJDE E A M=I HA =JA@ J =HCE E A?= A =I = IKHFHEIA 9A J =@L= J=CA B JDA B=?J JD=J =HCE E A EI = EJH CA I KH?A B H * IK>JE EI J ANF HAJDA=BBE EJO BJDEIFKJ=JELAFAH A=IA B H =HCE E AK @AHE LEL? @EJE I /H MJD =HCE E A=IJDA EJH CA I KH?A M=I E F=EHA@ E = B JDA JDHAA KJ= JI OGE: OGE; HOGE ECKHA = @@=J= JID M 6 AL= K =JAJDAABBE?EA?O BJDAFAH A=IA MA? F=HA@CH MJD E?HA=IE C=HCE E A??A JH=JE I ECKHA 6DAFAH A=IA = MI?A IJ CH M=JD= BJDA =NE K CH MJDH=JAMDA =HCE E AEI=J=??A JH=JE B! E JDAIAE LEL? @EJE I 6DEI FAH JDAHAB HA? @AIB H=DECD =BBE EJO=HCE E A FAH A=IA IE E =H J JDA =HJ213 FAH B -I?DAHE?DE=? E % 1? JH=IJ JDA=BBE EJO BJDAHAIE@K= FAH A=IAIEI K?D MAH BJDA H@AH B# ECKHA Methionine sulfoxide metabolism: the ytmijklmhispytmoytnijribr operon 6DA AJ=> EI B AJDE E A= @ 64M=IBKHJDAHE LAIJE C=JA@ *A?=KIAJDAFAH A=IAB H 64EIK M = >LE KI?= @E@=JAM=IJDAFAH A=IAA? @A@>OCA AIOJ DEI2 /H MJD B KJ= JI E JDAIA CA AI 64 M=I K =BBA?JA@ D MALAH@=J= JID M )IE JDA?=IA BOGE:;? F=HE I MEJD M CA AIE @E?=JA@JD=JJDACA AI? @AB H=FAH A=IA E A O J JH= IF HJ = E =?E@I H JDAEH = = CI 6DA BEHIJCA AE JDEI FAH OJ 1 EI E A OJ? @AB H= =?AJO =IA A?= ANFA?J JDAHAB HA JD=JJDAJH= IF HJA@IK>IJH=JA EI=?AJO =JA@>AB HA>AE C AJ=> E A@ 6DEIIKCCAIJIJD=JJDA IK>IJH=JA D=I J >A @ELAHJA@ BH = IJH=ECDJB HM=H@ E L LA A JE =FH?AIIMDAHAEJM K @>A@A AJAHE KI 5AGKA?A= EC A JI BJDAFH @K?JI BCA AIJD=J=HA=@ =?A J J JDA FAH A=IA CA A E JDA FAH KIE C FH JAE IAGKA?AI E @=J=>=IAI @=J= J ID M IKCCAIJA@ JD=J ;J ;J 1 4E>4 EI = @AFA @A J NOCA =IA =?JE C = IK BKH? J=E E C IK>IJH=JA " # 6DEI A O A? K @>AE L LA@E I AIJAF B 64@ACH=@=JE /H MJD 64 M=I D MALAH K =BBA?JA@ E JDA? HHAIF @E C KJ= JI @=J= J ID M 9A JDAHAB HA =JJA FJA@ J IAA MDAJDAH JDEI FAH M=I E L LA@ E JDA @ACH=@=JE B AJDE E A = @ AJDE E A IK B NE@A FH?AIIAI JD=J I?=L A CA IK BKH = @ FHALA J AJDE E A IK B NE@A CAJJE C E J FH JAE IO JDAIEI /H MJD BME @ JOFA>=?JAHE= AJDE E A IK B NE@A M=I >AJJAH JD= AJDE E A = =HCAH CH MJD HACE BH = @EI E FHAC =JA@ MEJD µ B = I KJE IKCCAIJE C JD=J JDA B H AH EI E @AA@ =? IK>IJH=JA E JDA A LEH A J /H MJD M=I O I ECDJ O =BBA?JA@ E JDA KJ= JI OJ = @ OJ MDA JDAO MAHA CH M E JDA FHAIA?A B = DECD??A JH=JE B AJDE E AIK B NE@A E = O CH MJDM=I >IAHLA@MEJD AJDE E AIK B A AEJDAHE JDAME @JOFA HJDA KJ= JI AN? K@E CJDEIIK>IJ=?A=I= E JAH A@E=JAE @ACH=@=JE @=J= JID M 9AJDAHAB HAE LAIJEC=JA@JDAIEJK=JE =J M??A JH=JE I BAEJDAH AJDE E A H AJDE E AIK B NE@A J O MEJD JDA FAH A=IA CA A KJ= JI >KJ = I comment reviews reports deposited research refereed research interactions information

4 Genome Biology Vol 2 No 6 5A MI =AJ= Table 2 Expression of arginine operons when cells were grown with methionine or with MTR as the sulfur source 10 WT yqix Gene and spot Met MTR Ratio argb a 1286 907 1.43 argb b 1329 928 argc a 1116 735 1.61 argc b 1145 666 argd a 930 592 1.62 argd b 1014 605 arge a 340 246 1.27 arge b 446 372 argf a 699 406 1.77 argf b 717 392 argg a 2304 1896 1.28 argg b 2827 2121 argh a 2165 1120 1.94 argh b 2237 1147 argj a 1045 509 1.94 argj b 1051 569 args a 991 838 1.16 args b 984 858 cara a 2329 2046 1.12 cara b 2326 2094 carb a 932 635 1.47 carb b 912 618 yqze a 680 460 1.57 yqze b 685 407 yqix a 2190 1401 1.58 yqix b 2264 1412 yqiy a 750 585 1.41 yqiy b 876 571 yqiz a 1527 926 1.70 yqiz b 1595 907 cdna hybridization after growth in methionine or MTR. The conditions are chosen to display roughly identical values for most genes in both conditions. The values obtained for the arginine operons described in the text are shown here. They indicate a higher expression in methionine as compared to MTR, all things being otherwise equal. Each spot in the experiment is referred to as a and b. MEJD= KJ= J BCA A OJ )IID M E ECKHA KJ=JE I E JDAIA CA AI IEC EBE?= J O E F=EHA@ CH MJD > JD JDAIA IK>IJH=JAI ID ME CJD=JJDAFAH A=IAEI=JH= IF HJIOIJA B HJDAIA AJ=> EJAI = @IKCCAIJE CJD=J AJDE E A@ACH= @=JE FH?AA@I =J A=IJE F=HJ JDH KCD NOCA =JE OD 600nm 1 0.1 0.01 0.001 0.01 0.1 1 10 100 Arginine concentration (mm) Figure 1 Comparison of the growth of B. subtilis wild type (WT) and yqix mutant (BFS4740) in different concentrations of arginine as the nitrogen source in glucose minimal medium (from 10 µm to 20 mm; note that arginine is toxic at 20 mm). Both strains were grown at 37 C with agitation (180 rpm). The OD was taken for both strains after 8 h growth, when they entered stationary phase (due to nitrogen limitation, except for the highest arginine concentrations). 6DEI >IAHL=JE M=I BKHJDAH IK>IJ= JE=JA@ >O JDA >IAHL= JE JD=JJDA KJ= JIMAHA HAHAIEIJ= JJ IA A AJDE E AJD= JDAME @JOFA@=J= JID M Discussion 1 IJ>=?JAHE= IK BKH AJ=> EI D=I J>AA IJK@EA@E =I K?D@AFJD=I?=H> EJH CA HFD IFD HKI AJ=> EI KHHA?A J= = OIEI BJDA@EIJHE>KJE BIK BKH AJ=> EI CA AIE -? E ID MA@JD=JJDAO=HA? KIJAHA@J CAJDAH E J EI = @I & 6DEI E @E?=JAI JD=J IA A?JE FHAIIKHA D=I B H?A@ JDA? HHAIF @E C CA AI J CH KF J CAJDAH = @ H JD=J JDAO D=LA HA =E A@ CH KFA@ J CAJDAH BH LAHO A=H O JE AI E JDA DEIJ HO B EBA 5K BKH HA?O? E C E F=HJE?K =H M=IB K @J F =O= E F HJ= JH AE?A K =HIK BKH AJ=> EI MEJD AJDO JDE =@A IE A 6) >AE C E F HJ= J JDEIEI@AHELA@BH = O?A HA=?JE I= @E F=HJE?K =HJDA IO JDAIEI B F O= E AI " 6DA E A@E=JA @ACH=@=JE FH @K?J B 6) 64 EI JHA?O? A@E -? E >KJEIABBE?EA J OHA?O? A@E * IK>JE EI FHAIK => O=I AJDE E A KH FHA E E =HO ANFAHE A JI ID MA@ JD=J?A I CH ME C 64 =I JDA IK BKH I KH?A >AD=LA@ =I @E@?A I CH ME C AJDE E A 6DEI IKCCAIJA@ JD=J O = I = IAJ B CA AI M=IE L LA@E JDAHA?O? E CFH?AII 9 K @EJ>AF IIE> AJ E@A JEBOI A BJDA 9A D=LA KIA@ ANFHAIIE FH BE E C J E@A JEBO JD IA CA AI JD=J=HA@EBBAHA JE= OANFHAIIA@MDA AEJDAH 64 H AJDE E A EI KIA@ =I JDA IK BKH I KH?A J J FH LE@A = C >=

http://genomebiology.com/2001/2/6/research/0019.5 Percentage of wild type growth 120 100 80 60 40 20 0 WT Met ytnj Met ytmj Met ytmk Met Met or MetO (100µM) WT MetO ytnj MetO ytmj MetO ytmk MetO Figure 2 Comparison of the growth of B. subtilis wild type (WT) with ytnj, ytmj and ytmk mutants (BFS70, BFS85 and BFS86, respectively) on methionine (Met) and methionine sulfoxide (MetO) at 100 µm concentration. Growth was measured after 7 h and is represented as a percentage of wild-type growth. FH BE AMDE?DM K @ KIJ>AKIA@B H@E=C IJE?FKHF IAI 1 FHE?EF A ANFHAIIE FH BE E C= MI AJ E LAIJEC=JAJDA? A?JELA>AD=LE H B=CH KF BCA AI BE JAHAIJ>KHEA@E JDABK CA A 9DA JDAHAEI K?DK? JH A@L=HE=JE E JDA KJ? A B DO>HE@E =JE D MALAH EJ >A? AI @EBBE?K JJ @EIJE CKEIDH= @ B K?JK=JE BH =IFA?EBE?ABBA?J 6DA M H FHAIA JA@ DAHA M=I E JA @A@ J E@A JEBO =J A=IJ I A B JDA?=KIAI B L=HE=JE E ANFHAIIE FH BE E C E >=?JAHE= IE F A B K?JK=JE I = @ IOIJA =JE? >E=IAI JHOE C J ANJH=?J BH CA AH= >=? CH K @ EIA JD IA BAM CA AI JD=J@EIF =O@EBBAHA JE= ANFHAIIE 6DAB=?JJD=JMA?= AKF MEJD=I K @>E CE?= ANF = =JE BJDAL=HE=JE I@A IJH=JAIJD=J KHANFAHE A J= = @IJ=JEIJE?= M H F = EIABBE?EA J F=LE C JDA M=O B H BKHJDAH HA? F AN HAIA=H?D KIE CJH= I?HEFJ A= = OIEI Statistics 9AD=LA?D IA J KIAL=HE=?A= = OIEI =IJ=JEIJE?= =FFH =?D JD=J D=I C >AA KIA@ >O A? EIJI = @ BEA @ I?EA JEIJI MD D=LAJ @A= MEJD= =HCA K >AH BFEA?AI B@=J=K @AH? @EJE I MDAHA JDA? JH B JDA A LEH A J EI F H 6DAIAJA?D EGKAID=LA>AA L= E@=JA@JDH KCD HAJD= $ OA=HI B D=H@ M H = @ MA JDE JDAO ID K @ >A HA BHA GKA J O? IE@AHA@ B H =HCA I?= A CA E?I ANFAHE A JI JDAHJA?D EGKAI IK?D=IH= = = OIEI =HA= I HA AL= J >KJ L=HE=?A = = OIEI EI KIABK B H = = O E C JDA =JKHA B IOI JA =JE? HH= @ AHH HI=BBA?JE C@=J= MDA? IE@AHE C= =HCA K >AH B@=J=F E JI 6DA>=IEI BJDAIAJA?D EGKAIEIJD=J@=J=JHA=J A JFHE HJ = = OIEI KIJ>AHA@K?A@J = E E K B HAN= F A A ID K @ JD=LAJ IK>JH=?J>=? CH K @L= KAI HKIAH=JE A=IKHA A JI >A?=KIAJDEI?= O>A@ AMEJD KJE JH @K?E C IFA?EBE? >E=I K @AH DECD O? IJH=E A@ IJ=JEIJE?=? @EJE I MDE?D =HA?AHJ=E O J BK BE A@ E JH= I?HEF J A ANFAHE A JI 5E E =H O A ID K @ J E JH @K?A H = E =JE? @EJE I >=IA@ K @AH OE C >E CE?= DOF JDAIAIB HAN= F A KIE CJDA=LAH=CA BI AD KIA AAFE CCA AI MDE?D=HAIKFF IA@J >AANFHAIIA@=J=? IJ= J ALA >A?=KIAJDEIE JH @K?AI=IOIJA =JE?>E=I MEJD JDA=IIK FJE JD=J B HJM CELA CA AJE? HA LEH A J=? @EJE I JDAANFHAIIE ALA BIK?D? JH CA AIM K @ JL=HO = OANFAHE A JI ' ID MJD=JJDEIEI?AHJ=E O JJHKAE CA AH= 6DA KJ? A B JDA L=HE=?A = = OIEI EI J E@A JEBO JD IA CA AI JD=J L=HO E =? IEIJA J = AH B H = CELA ANFAHE A J= IAJ KF?A JDAIA CA AI D=LA >AA E@A JEBEA@ B? KHIA EJ>A? AI A?AII=HOJ = A=GK= JEJ=JELAAL= K= JE B JH= I?HEFJE = @ JH= I =JE E JDA? @EJE I B E JAHAIJJ >AIKHAJD=JJDAO=HAE @AA@HA AL= J Preparation of the cdna *=?JAHE= ANFHAIIE FH BE E CEIF=HJE?K =H O@EBBE?K JJ IAJKF >A?=KIA E? JH=IJ J AK =HO JAI >=?JAHE= @ J = A IJ=> AF O=@A O =JA@ 4 ) 1 B=?J JDA=LAH=CAJKH LAH B 4 ) K @AH KIK= CH MJD? @EJE I B H @A HC= EI I IK?D=I-? E = @* IK>JE EI EI BJDA H@AH B E KJAI )I ID M E KH= = OIEI ANFAHE A J= >E=IAI EA=JJDA ALA B 4 ) HA? LAHO = @ IO JDAIEI B = =>A A@?, )? FO B H DO>HE@E =JE J JDA, )=HH=OI 6DA= K J B?, )IO JDAIE A@EI JFH F HJE = J JDA= K J B4 )= @M HIA JDAOEA @ B?, )IO JDAIEI=FFA=HIJ L=HOBH CA AJ CA A =LAH=CA= K JI B 4 )IFA?EAICELE CJDA IJL=HE=> A?, ) KJ? A 1 CA AH= JDAHA=I CELA B HKIE C =HCAH = K JI B 4 ) EI J =L E@ JDA IEJK=JE MDAHA JDA MAIJ ANFHAIIE ALA I=HADE@@A E JDA>=? CH K @ EIA 1? JH=IJ EJ EI JDA BA=H JD=J JDA A >H= A M K @ >A I=JKH=JA@ = @JDKI A F=H@E AJDA=??KH=?O BJDAL= KAI? HHAIF @E C J JDA DECDAIJ ANFHAIIE ALA I JD=J CKE@AI JDA?D E?A B = I = = K J B4 )E I AANFAHE A JI 9AAL= K=JA@JDAE F HJ=?A BJDA>E=IAI@KAJ JDA>=? CH K @ EIA JDA AD= @= @J JDAI=JKH=JE FH > A JDA JDAH>OIJK@OE CJDA? HHA =JE IE A=IKHA A JI FAHB H A@ KIE C AEJDAH µc H µc 4 ) BH JDA I= A ANJH=?J ) II BIA IEJELEJO@KAJ JDA>=? CH K @ EIAME = EBAIJEJIA B=I= M? HHA =JE >AJMAA JDAH= H@AH B JDACA AIE A=?DANFAHE A JCA AID=LE C>AA H= A@=I= BK?JE B JDAEH IEC = E JA IEJO B H JDA CA AI CELE C M = K JI B?, ) = @? LAHIA O I=JKH=JE ME A=@J = comment reviews reports deposited research refereed research interactions information

6 Genome Biology Vol 2 No 6 5A MI =AJ= F H? HHA =JE B H CA AI CELE C =HCAH = K JI B?, ) A BJDAIA>E=IAID=@=IEC EBE?= JE B KA?AE KHANFAH E A JI 1 JDA=>IA?A BIK?D>E=I JDAH= E C BJDACA AI EIJA@=I=BK?JE BJDAIEC = E JA IEJO KIJ>AJDAI= A E A=?D ANFAHE A J 6DEI EI J MD=J D=I >AA >IAHLA@ AN?AFJ=JJDA M= @DECDA @I BJDAI?= A ) ANF = =JE B H JDEI =? B? HHA =JE D=I >AA FH F IA@ E I A CA AI?, ) IO JDAIEI =O >A =BBA?JA@ >O JDAB H =JE BIA? @=HOIJHK?JKHAIE JDA 4 ) H>OJDA FHAIA?A B4 )IAC A JI BDECD O>E=IA@ K? A JE@A? F IEJE 6DAIA =HA? @EJE I MDAHA JDA ABBE?EA?O B?, )IO JDAIEI ECDJ>A=BBA?JA@B H =HCA4 )??A JH= JE I )JD H KCDIJK@O BJDAIAGKA?A BCA AICELE CJDA IJAHH=JE?DO>HE@E =JE L= KAI =O= M AJ AIJE =JA JDAL= KA BJDEIDOF JDAIEI IJH=ECDJB HM=H@HA=I? K @ >A B K @ E JDA FHAIA J ANFAHE A JI )I @A IJH=JA@ >O 0=JBEA @= @? M H AHI JDAKJE E =JE BH= @ DAN= K? A JE@A FHE AHI ID K @ = ALE=JA I A B JDEI @EBBE?K JO = A O 4 )B @E C A=HJDA! A @ BJDA 4 ) 6DAEH M H @E@ J D MALAH ANF HAE @AJ=E IJDAABBA?J B4 ) GK= JEJEAI JDA KJ? A BJDADO>HE@E =JE F=JJAH = @ EJ =O MA >A JD=J >E=IAI IE E =H J JD IA @AI?HE>A@ DAHA M K @ >A K? LAHA@ E = O JDA H A B? FAJEJE B = =HCA K >AH BFHE AHI B@EBBAHA JIAGKA?AI =IMA =I B JDA =HCA GK= JEJO B H4 ) FHAIA J E JDA?, ) HA=?JE ENJKHA D=I JOAJ>AA = = O A@E @AFJD KHJDAHM H EI AA@A@ >AB HA MA M JDA HA =JELA? JHE>KJE I B JDAIA F=H= AJAHI = @ EJ?= >A ANFA?JA@ JD=J E JDA A=H BKJKHA =FFH FHE=JAFH J? IME = ALE=JAI A BJDAIADOF JDAJE?= >KJF =KIE> A?=KIAI B>E=I Growth culture conditions 6DAANFAHE A JI@AI?HE>A@DAHA= MA@KIJ FHE HEJE AJDA I KH?AI B>E=IAI= @=HJEB=?JIE JDA= = OIEI BJDA>=?JAHE= JH= I?HEFJ A 1 =@@EJE J? JH E C?, )IO JDAIEI MA B K @ JD=J? JH E C?K JKHA? @EJE I M=I HA E F H J= J JD= ANFA?JA@ 7 =FFHA?E=JA@ @EBBAHA?AI E?K JKHA? @EJE IIAA J HAIK JE? H@E =JA@L=HE=JE I BCA A ANFHAIIE 1 @AA@ MA >IAHLA@@EBBAHA?AIE JDAANFHAIIE B? FA JA?A FAH I @EBBAHA J @=OI 6DEI E @E?=JAI JD=J IK>J A @EBBAHA?AI E CH MJD? @EJE I HAIK J E GKEJA LEIE> A = @ IA B? IEIJA J @EBBAHA?AI E CA A ANFHAIIE + FAJA?A =IMA =IIF HK =JE EI EJ HA@>OJDA?A KIE CIK>J A?KAI IJ B MDE?D =HA IJE K M 6DEI ANFAHE A J ID MIJD=JJDAIAFH?AIIAI=HAANJHA A OIA IEJELAJ CH MJD? @EJE IANFAHE A JID=LA>AA HAFH @K?A@=IAN=?J O=I F IIE> A E? K@E C? JH B =CEJ=JE EJ HE C CH MJD = @JHOE CJ AAFALAHOJDE C=I? IJ= J=IF IIE> A 6DEI M=H I =C=E IJ FHA =JKHA =IIEC A J B ABBA?JI? FA JA?A HIF HK =JE FH?AIIAIBH ANFAHE A JIMDE?DD=LA J>AA IJ=JEIJE?= O? JH A@ 1JEI? A=HJD=JIE F O?=HHO E C KJ @EBBAHA JE= ANFHAIIE ANFAHE A JI M K @ D=LA A@ KI J E JAHFHAJ = ABBA?J JD=J EI IE F O @KA J JDA ANJHA A IA IEJELEJO BJDEIFH?AIIJ IK>J A?D= CAIE A LEH A J=? @EJE I =I = ABBA?J B 64? FAJA?A IAA JDA? CA AI E 6=> AI! 2HALE KI HAIK JI JDAIA E JAHAIJE CFDA A =ID K @>AHA? IE@AHA@E JDEI ECDJ -LAHO>E CEIJ MID M@EBBE?K J= @JE A? IK E CEJEI J HAFH @K?A AN=?J O E = DECD O GK= JEJ=JELA = AH J O JDA ANFAHE A JI FAHB H A@ E = JDAH => H=J HO >KJ = I JD IAFAHB H A@E IE@A A\I M => H=J HO 1 @AA@ EJ EI H=HA JD=J = ANFAHE A J=? @EJE I?= >A? JH A@ BE A O A KCD J = M B H = HEC H KI O AN=?J HAFAJEJE @AIFEJA E FHAIIE I J JDA? JH=HO H AN= F A E BK? JE = >=?JAHE= CA E?IANFAHE A JI A@ AI JANF E? EJ O? JH MD=JD=FFA IJ JDA?A I>AJMAA FHA?K JKHA= @ CH MJDE JDABE = A@EK ) JD KCDJDAIAJM IJAFI??KH KIK= O=J!% + IJ BJA >=?JAHE==HAF =?A@B H=L=HE=> A JE A E = H MDAHA JA FAH=JKHA EI IEC EBE?= J O MAH = @ KIK= O J? JH A@ = O AFECA AJE? FDA A = =HAJHECCAHA@@KHE CJDEIJH= IEJE FAHE @ = @JDEI =O A=@ J @EBBAHA?AI E CA A ANFHAIIE 6DAIA @EBBAHA?AI ME >A H= @ >KJME? HHAIF @J K? JH A@@EBBAHA?AI >AJMAA ANFAHE A JIJD KCDJJ >AJ J= OE@A JE?= 6 CELA KIJ AAN= F A = OANFAHE A JI=HAFAHB H A@=J[H JA FAH=JKHA\ MDE?DEI=LAHOF H O@ABE A@FDOIE?= F=H= AJAH 6DAHA =HA?AHJ=E O JDAH B=?J HI IAA B H AN= F A JDAH A BC =IIM=HA?=H> @E NE@A= @JDAGK= EJO B JDAM=JAHMDE?D=HA@EBBE?K JJ? JH = @ = AJDA= = O IEI BJDAANFAHE A JI CA AANFHAIIE @EBBE?K J Methionine and methylthioribose (MTR) 6DA ANEIJA?A B =HCA E JAH=?JE I B JDA JOFA CA A? @E JE E EJIJDAE B H =JE JD=J?= >AANJH=?JA@BH =JH= I?HEFJ A = = OIEI 6DA IJ=JEIJE?= = = OIEI D MALAH A => A@KIJ E@A JEBOCA AIJD=JMAHA= IJ?AHJ=E O=II?E=JA@MEJDCELA FDOIE CE?=? @EJE I 64 EI ABBE?EA J O HA?O? A@ E * IK>JE EI KIE C = OAJ K M F=JDM=O 1J IAA I HA=I => A J E BAH JD=J JDEI HA?O? E C EI JDH KCD AJDE E A =I E A>IEA = F AK E=A? K JAHF=HJI BJDA? HHAIF @E C F AK E=A CA AI IAA J >A FHAIA J E * IK>JE EI D MALAH 6DA FHAIA J ANFAHE A J M=I JDAHAB HA E JA @A@ J E@A JEBO JDA BEHIJ A A A J B JDA F=JDM=O AJDO JDE HE> IA FAH A=IA = @ HJDACA AIHAIF IE> AB HEJIHA?O? E C >O? F=HE C CH MJD 64 MEJD JD=J AJDE E A EJI E A@E=JA @AHEL=JELA 6DA? HHAIF @E CCA AIID K @E @AA@IJ= @ KJ EB JDAEH ANFHAIIE EI E @K?E> A >O 64 =JKH= O >A?=KIA 64 EI = H = FH @K?J B JDA?A JDA? HHAIF @E C CA AI ECDJ>AANFHAIIA@? IJEJKJELA O E MDE?D?=IAJDAO ME J>AE@A JEBEA@>OJDEIFH?A@KHA IEC EBE?= JE @K?JE M=I >IAHLA@ >KJI AHAFHAIIE BJDA=HCE E A>E IO JDAJE?F=JDM=OM=I? IEIJA J OB K @ E CH MJD 64 6DEI E JAHAIJE C E >AJMAA EJH CA = @ IK BKH AJ=> EI =O >A =?? K JA@ B H = @ = AI 64 H A B EJI E A@E=JA @AHEL=JELAI = E JAHAIJE C

http://genomebiology.com/2001/2/6/research/0019.7 Table 3 Function of genes showing the largest differences between days A and B Gene Mean Standard Function deviation comea 2.588 0.930 Late competence protein required for comfa 3.213 0.809 Late competence protein required for DNA uptake comfc 2.448 0.713 Late competence protein required for DNA uptake comga 4.794 0.956 Late competence operon required for comgb 3.649 0.604 Late competence operon required for comgc 2.644 0.724 Late competence operon required for comgd 3.512 0.828 Late competence operon required for comge 4.067 1.038 Late competence operon required for nuca 3.134 0.954 Competence-specific nuclease raph 3.453 0.619 Response regulator: aspartate phosphatase (sporulation) ypze 2.261 0.143 Hypothetical protein yqze 3.856 0.810 Hypothetical protein ysha 2.331 0.718 Conserved hypothetical protein yvrp 2.697 0.966 Conserved extracytoplasmic component of a permease (hypothetical) cotg -2.547 0.784 Spore coat protein glga -2.335 1.053 Bacterial glycogen synthase rplv -2.465 0.958 Ribosomal protein L20 ybfo -2.884 0.880 Similar to erythromycin esterase (detoxification) ylla -3.531 0.914 Hypothetical protein yojl -2.631 0.710 Similar to cell wall-binding protein?= @E@=JA B H =? HAFHAII H EB A JAI JD=J =HCE E A EI JDAFHA?KHI H BF O= E AIE * IK>JE EI= @JD=J 6) JDA E A@E=JAFHA?KHI H B 64 EIFH @K?A@E =IJ E?DE AJHE? B=IDE MDA JDAF O= E AIFAH E@E AEIIO JDAIE A@ " 1J =O >A HA AL= J J HA =H JD=J JDA EI =JA@ =HC- CA A EI FHAIA J E = FAH B K M BK?JE MEJD IALAH= CA AI D=LE C IEC EBE?= J D CO MEJD CA AI E L LA@ E IK BKH AJ=> EI HA LAH JDA OJ, CA A E JDA =HC/0OJ, FAH A? @AI = IAGKA?A HE?D E IK BKH? J=E E C= E =?E@I MDE?DEIK KIK= E * IK>JE EI FH LE@ E C = BKHJDAH E @E?=JE B = IEC EBE?= J? KF E C >AJMAA =HCE E A= @IK BKH AJ=> EI E JDEI HC= EI 1 =@@EJE ANFHAIIE BJM FAH IA? @E CFAH A=IAI OGE:; = @OJ DEI2L=HEAIE FF IEJAM=OIMDA?A I=HACH M AJDE E A H 64 6DEIFH FJA@KI J A=IKHAJDACH MJD B KJ= JI BA=?D BJDAIACA AI L=HE KI IK BKH I KH?AI IEC EBE?= J CH MJD E F=EH A J M=I >IAHLA@ MDA??A JH=JE B JDA AJ=> EJAI M=I DECD 6DA IJ F =KIE> A ANF = =JE EI JD=J 64 = @ AJDE E A?= >AJH= IF HJA@E J JDA?A >OIALAH= FAH A=IAI FAH A=IAI JH= IF HJE C AJDE E A =HA = HA=@O M I JD=JE =?JEL=JE B A BJDA @ AI J= JAH CH MJD KHJDAHM H ID MA@JD=JJDAOGE:; CA AFH @K?J M=IJH= IF HJE C=HCE E A 6 >A? IEIJA JMEJDJDA-? E A? =JKHA IKFF HJA@ >O 5MEII2H J MA FH F IA J HA = A JDAIA CA AI =HJ23 B H =HCE E A JH= IF HJ 6DA A?K =H =JKHA B JDA E >AJMAA IK BKH = @ =HCE E A HA =E IJ >AE LAIJEC=JA@ D MALAH )IE E =HE L LA A J E JH= IF HJ M=I MEJ AIIA@ MEJD JDA OJ DEI2 CA AI 6DAIACA AI=FFA=HJ? @AB H=FAH A=IAB H AJDE E A = @ AJDE E A IK B NE@A = @ JDA FAH EI A= J J @ACH=@A AJDE E A>O= @AFA @A J NOCA =IA FHAIK => O =BJAH =?AJO =JE 5 MA D=LA JA J=JELA O HA = A@ JDA CA AI OJ 1 OJ DEI2 OJ OJ 1 = @ HE>4HAIFA?JELA O=I @C) @C 2365 @C AJDE E A IK B NE@A @ACH=@=JE FAH A=IA = @ JH= IF HJ N)*+ N AJDE E A IK B NE@A NE@=IA = @ H>B H>B HE> B =LE E =IA JDKI=L E@E C[HE>\ MDE?DKIK= OIJ= @I B HHE> IA 6DAANEIJA?A BJDEI FAH EIA=IOJ =?? K J B H ME C JD=J JDA KIK= >E J FA B * IK>JE EI EI JDA FDO F = A 1 @AA@ EB AJDE E A EI ANK@A@ BH F = J A=LAI JDA JDEI??KHI E JDA FHAIA?A B = DECD ALA B @E NOCA MDE?DME ABBE?EA J O AJ=> E AJDA AJDE E A J AJDE E AIK B NE@A @EBE?=JE F IIE> O=?AJO =JE BJDAIK>IJH=JAME FHALA JEJBH CAJJE CE J JDAFH JAE IO JDAIEIF=JDM=O E = O E @K?JE B CA AI E L LA@ E I?=LA CE C NOCA H=@E?= IBKHJDAHIKCCAIJIJD=J=FH?AIIE L LE C NOCA EI?=HHEA@ KJE JDAHA?O? E C B 64 6DEIEIE E AMEJDJDA E L LA A J B=@E NOCA =IAE 64HA?O? E CE F AK E=A! Materials and methods Bacterial strains and growth conditions 6DAB ME C* IK>JE EIIJH=E IMAHAKIA@E JDEIM H JDA ME @ JOFA IJH=E $& " * 5% OJ =? * 5&# OJ =? * 5&$ OJ =? * 5"%" OGE: =? * 5"%!' OGE; =? = @ * 5"%!& OGE =? 6DAIA IJH=E I D=LA >AA? IJHK?JA@ MEJDE JDA -+ FH A?J B H JDA BK?JE =?D=H=?JAHE =JE B JDA CA A B * IK>JE EI E -KH FA* 5% * 5&# * 5&$ # = @E =F= * 5"%" * 5"%!' * 5"%!& $ *=?JAHE= MAHA CH M E -, E E = A@EK 02 " & 0 2 " " " C K? IA % =!?EJH=JA! C KJ= E A # JHOFJ FD= "" BAHHE??EJH=JA!! # µ C+ $ +=+ "' # µ A+! "' ' µ + # # µ comment reviews reports deposited research refereed research interactions information

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10 Genome Biology Vol 2 No 6 5A MI =AJ= 6DA@EBBE?K JEAI>ACE MDA MABE @E JAH=?JE I>AJMAA JM B=?J HI = @ CA AI CA A N @=JA N IK BKH I KH?A CA A N = K J B4 )NIK BKHI KH?A = @CA AN@=JAN= K J B 4 )>A?=KIAJDEIE @E?=JAIJD=JJM B=?J HI=?JIE K J= A KI O JDA L= KAI A=IKHA@ B H I A CA AI 1 JDAH M H@I A?=? FKJA JDA @EBBAHA?A >AJMAA JM A= IKHA A JI JDAI= ACA A >KJEJEI JF IIE> AJ =IIEC JDA L= KA B JDEI @EBBAHA?A J JDA I A ABBA?J B?D= CAI E? @EJE I >AJMAA JDA JM A=IKHA A JI 6=> AI EIJE C HAIK JI B JDEI JOFA KIJ JDAHAB HA >A E JAHFHAJA@ MEJD ANJHA A?=KJE EJ M K @ >A ALA M HIA B? KHIA EB A KIA@H=JE I =IEIK B HJK =JA O BJA @ A )FHK@A JI K JE EIJ??A JH=JA JDACA AIJD=J@ JID ME F H J= JL=HE=JE I AN?AFJB H A BJDAB=?J HI 1 FH=?JE?A A? FKJAIB HA=?DCA AJDA@EBBAHA?A B A=IKHA A JIB H= CELA B=?J HB HAN= F AJDAIK BKHI KH?AE JDAB KH? @EJE I HµC @=O) H* JDA A?=?K =JAIJDA A= = @JDAIJ= @=H@@ALE=JE B= B KH@EBBAHA?AI 6DA IJ E JAHAIJE C CA AI =HA JD IA JD=J? >E A JDA =HCAIJ A= = @ JDA MAIJ IJ= @=H@ @ALE=JE 6DA CA AH= I KJE J JDAFH > A ALA MDA JDAB=?J H BE JAHAIJD=I HAJD= JM L=HE=> AIB HAN= F AJDAIE K J= A KIIJK@O BJDHAA H HA IK BKH I KH?AI EI J FAHB H B H A=?D CA A = L=HE =?A= = OIEIMEJDJM B=?J HIJDAB=?J H BE JAHAIJ JDA A D= @ = @ = JDA JDAH AI CH KFA@ J CAJDAH B H AN= F A @=JA 4 )= K J JDA JDAH JDA J HAJ=E JD IA CA AI JD=J @EIF =O > JD = MA= E JAH=?JE >AJMAA B=?J HI= @= =HCAIK BJDAIGK=HAI BJDA@ALE=JE IBH JDA A= B HJDAB=?J H BE JAHAIJ Controlled factors A?= HA=I => O=IIK AJD=JJDA >IAHLA@DO>HE@E =JE IEC = EI FH F HJE = J JDA?, ) = K J IF JJA@ >O JDA H > J =I JDA A=IKHA A J B JH= I?HEFJE ANFHAIIE EI >=IA@ JDA DO>HE@E =JE B? F AN FH >AI =J M + J K @AH? @EJE I MDAHA JDA J=HCAJ IAGKA?AI =HA E =HCA AN?AII JD=J EI B=H BH JDA I=JKH=JE JDHAID @ 9A D=LA J= A BELA B=?J HI E J =?? K J EHIJ JDA? @EJE B JDACA AANFHAIIE @A JA@IK BKHI KH?A MEJDJM ALA I 5A? @ JDA?K JKHA @=O B 4 ) ANFAHE A JI MEJD I= A =JAHE= = @ FH J? MAHA =@A =J JM @EBBAHA J @=OI 6DEH@ JDA= K J B4 ) µc HµC KHJD JDAH > J =OI JM IK??AIIELA @H FI JDA A >H= A ANJ J A=?D JDAH JDEIB=?J HEI@A JA@@H FMEJDJM ALA I = @ -NFAHE A JI MEJD ALA = @ =HA?= A@ HAF E?=JAI EBJD JDACA A JDAHA=HA" %CA AI 9A JDKI D=LA " N " % $# % JHA=J A J K EJI -NFAHE A JID=LA>AA =@AMEJDJM A >H= AI 6DA A >H= A B=?J H EI IKFAHE F IA@ JDA E JAH=?JE @=O N = K J B 4 )ANJH=?JE I MA=HA J=> AJ @EIJE CKEIDJDAIAJM I KH?AI BL=HE=JE 6=> A "ID MIJDA?D=H=?JAHEIJE?L= KAIB HJDADO>HE@E =JE E JA IEJO @EIJHE>KJE E JDA @EBBAHA J ANFAHE A JI 6DA BECKHAIB HA=?DCA A? HHAIF @J JDA=LAH=CA BJDAE JA IEJO BA=?D A BJDA@KF E?=JA@H FIE =H>EJH=HOK EJI JDA L= KACELA EIJD=J BJDA@H FMEJDJDADECDAIJL= KAE A=?D GK=HJE A 6DA@EIJHE>KJE EILAHO=IO AJHE?= 6DAIFHA=@ B JDA @EIJHE>KJE =I A=IKHA@ >O JDA @EBBAHA?A >AJMAA JDA BEHIJ = @ JDA JDEH@ GK=HJE A L=HEAI =I = BK?JE B JDA = K J B 4 ) KIA@ J IO JDAIE A?, ) µc "%' ± & µc %&#± # 6DAI= AD @IB HJDA=LAH=CAL= KA MEJD = B=?J H $ >AJMAA JDA JM FH J? I MDE A JDA 4 ) = K JKIA@L=HEA@>O=B=?J HµC #!± && µc %&% ±!$ 6DAHAANEIJI=? A=H@EBBAHA?A>AJMAA JDA A=IKHA A JIFAHB H A@KIE CµC4 ) @=OI)= @* 6DEI@EB BAHA?AEI J >IAHLA@E JDAFHAF=H=JE IFAHB H A@KIE C JDALAHOI= AANJH=?JI MEJDµC4 ) 8=HE=?A = = OIEI ID MI JD=J JDA E JAH=?JE I E L LE C JDA = K J B 4 ) =HA HA E F HJ= J JD= JD IA @KA J = JDAHB=?J HI 6D=JEI L=HE=JE IBH CA AJ CA A MEJDE = I= AANFAHE A J BJDAH=JE >AJMAA JDAGK= JEJO B?, ) = @JD=J B 4 ) =HA HAE F HJ= JJD= JDAL=HE=JE I HAIK JE CBH JDA?K JKHA@=O HJDAIK BKHI KH?A 6DEIEI D MALAH =B=?J H BJA AC A?JA@E ANFAHE A J= FH J? I = @JDAHAB HA@AIAHLAIIFA?E= IJK@O 6 JDEI A @ JDA CA AI MAHA H@AHA@ =I = BK?JE B JDA =LAH=CA E JA IEJO B JDA IEC = JDA @EIJHE>KJA@ E J BELA? =IIAI MEJD JDA I= A K >AH B A A A JI 6DA BEHIJ? =II?A JE AIBH J? J=E A@JDA&!CA AID=LE CJDA MAIJIEC = E JA IEJO A=IKHA@ JDAJ J= EJO BJDAAECDJ ANFAHE A JI + =II "? J=E A@ JDA IK>IAGKA J &! CA AI MDE?DD=LA=IEC = MEJD= E JA IEJODECDAHJD= JD=J Table 4 Characteristic values for the distribution of intensities (arbitrary units) in different experimental conditions Met A 1 MTR A 1 Met B 1 MTR B 1 Met A 10 MTR A 10 Met B 10 MTR B 10 First quartile 406 381 300 293 523 548 532 488 Second quartile 597 559 434 422 762 833 797 756 Third quartile 970 913 719 693 1,230 1,371 1,335 1,294 The genes were ranked according to the intensity of the corresponding hybridization; the values were then arranged into four sets of equal numbers, with increasing hybridization values. The value for the highest hybridization in each quartile is given here. Met, methionine; MTR, methylthioribose; A and B refer to different days, and 1 and 10 refer to the amount of RNA (see text).

http://genomebiology.com/2001/2/6/research/0019.11 B? =II = @ MAHJD= JD=J B? =II" $ = @I 6DA? HHA =JE >AJMAA JDAL= KAI >J=E A@KIE CµC= @ JD IAFAHB H A@KIE CµC B4 )M=I?=?K =JA@B HA=?D? =II6=> A # A >IAHLA@=C @? HHA =JE >AJMAA JDA A=IKHA A JIFAHB H A@MEJDµC= @µc B4 ) MDA JDAIEC = M=IAEJDAHLAHO M HLAHODECD 6DEIID MA@BEHIJ JD=J JDA AN?AII B IAGKA?A J=HCAJ, ) EI IKBBE?EA J B H DO>HE@E =JE J >AB=HBH I=JKH=JE = @IA? @ FAHD=FI K ANFA?JA@ O JD=J JDA >=? CH K @ EIA JDA HAIE@K= H=@E =?JELEJO BJDA A >H= A = @I @ AI J= JAHJDA A=IKHA A J BDO>HE@E =JE B M?, )= K JI 1? JH=IJ JDA? HHA =JE M=I LAHO F H B H JDA A@EK DO>HE@E =JE L= KAI 6 LAHEBOJD=JJDEIM=I J= =HJEB=?J @KAJ JDA MAHL=HE=?AE JDA?A JH=? =IIAI MA?=?K =JA@ JDA? HHA =JE >AJMAA JDAL= KAI >J=E A@KIE CJDAI= A = K J B4 )ANJH=?JA@BH =?K JKHACH M MEJD AJDE E A = @ = JDAH MEJD AJDO JDE HE> IA 6=> A $ 6DA Table 5 Variation of the correlation in the measurements performed with 1 µg and 10 µg RNA Centile Met A 1 x Met B 1 x MTR A 1 x MTR B 1 x Met A 10 Met B 10 MTR A 10 MTR B 10 0-20 0.589 0.589 0.563 0.668 20-40 0.132 0.248 0.103 0.422 40-60 0.018 0.087-0.022 0.332 60-80 0.191 0.179 0.120 0.350 80-100 0.698 0.763 0.706 0.807 The results for experiments with 1 or 10 µg RNA were compared after ordering the genes with the corresponding hybridization values in increasing order. A high correlation indicates that the results are very similar for both conditions. The correlation is very good for highly expressed or poorly expressed genes whereas it is low for those with medium expression. Met, methionine; MTR, methylthioribose; A and B refer to different days, and 1 and 10 refer to the amount of RNA (see text). Table 6 Variation of the correlation in the measurements performed with two sources of sulfur Centile Met A 1 x Met A 10 x Met B 1 x Met B 10 x MTR A 1 MTR A 10 MTR B 1 MTR B 10 0-20 0.751 0.767 0.636 0.777 20-40 0.685 0.680 0.514 0.681 40-60 0.730 0.663 0.608 0.749 60-80 0.821 0.736 0.738 0.843 80-100 0.945 0.912 0.946 0.969 The data are arranged as in Table 5. Correlation is good, indicating that there is not a large difference between growth on methionine and growth on MTR (as expected).? HHA =JE IMAHA K?DDECDAHJD= JD IAB K @E 6=> A # @AIFEJAJDAB=?JJD=JJDA? =IIAI? J=E A@AN=?J OJDAI= A CA AI = @JD=J A=IKHA A JIL=HEA@LAHO EJJ A B HJDAL=IJ = HEJO BJDACA AI BH AIK BKHI KH?AJ JDA JDAH 6DA =>IA?A B = C @? HHA =JE >AJMAA JDA A=IKHA A JI FAHB H A@ KIE C µc H µc 4 ) B H A@EK DO>HE@E =JE L= KAIEIJDKIHA= 1 JDAHJAH I JDAAIJE = JE BJDAANFHAIIE B=CA A@AFA @I? IE@AH=> O JDA = K J B 4 ) KIA@ J IO JDAIE A EJI?, )? F A A J @AIFEJAJDAB=?JJD=JMAKIA@JM = EGK JI B=K EGKA4 ) FHAF=H=JE 6DEIFDA A EIF=HJE?K =H OE F HJ= JB H JDA CA AI JD=J CELA = A@EK DO>HE@E =JE = @ JDAHAB HA JH= I?HEFJE IEC = HJDEIHA=I MA?= =IIAHJJD=JJDA >IAHL=JE I =@A E JDA FHAIA J IJK@O @H=M JDA E E =?? KIE IJD=J?= >AANJH=?JA@BH JDA@=J= = @JD=JEJEI E A OJD=JJDAHA=HABKHJDAHABBA?JI>KHEA@E JDA@=J= E F=H JE?K =HE L LE CCA AIMEJD A@EK ANFHAIIE )H=FE@>H MIAJDH KCDJDA EIJ BJDA IJL=HE=> ACA AI @E@ J HALA= = O IFA?E= BA=JKHA = A O JDAHA EI = FAH BA?J O H = HAFHAIA J=JE BK M [O\CA AI" =I? F=HA@J $' "E JDACA A= @JD IAMAHA JJDA I= ACA AI=IE JDAJM FHA?A@E C EIJI 6DA HAFA=J=>E EJO B JDA H > J IF JJE C EI C @ = @ JDEI I KH?A B =HJEB=?J?= JDAHAB HA >A @EIHAC=H@A@ '# B JDA CA AI @EIF =O = @EBBAHA?A >AJMAA > JD IF JI MAH JD= 1JEI JF IIE> AJ M MDA =IF JEIIOIJA =JE?= O HAE JA IAJD= EJI? K JAHF=HJ MDAJDAHJDA>E=IEI @KAJ I AIFA?EBE?BA=JKHA BJDACA A H@KAJ JDA A >H= A B H AN= F A EHHACK =HEJO B JDA H > J DAJAH CA AEJO BJDA>=? CH K @>A?=KIAA=?DCA A AAFIJDAI= AF IE JE E JDA A >H= AE = ANFAHE A JI Uncontrolled factors 6DA @EBBE?K JO B BE A O? JH E C JDA ANFAHE A J=? @E JE IBH A@=OJ JDA JDAHEIJDAIA? @I KH?A BIOI JA =JE? AHH HI 6DA EIJ B JDA IJ L=HE=> A CA AI =?? H@E CJ JDEIF=H= AJAHD=IIALAH= E JAHAIJE CBA=JKHAI /A AI B M BK?JE MAHA=> H = O=>K @= J! JDA @EBBAHA?A BH JDA FH F HJE E JDA CA A EI DECD O IEC EBE?= J F E @E?=JE C= H= @ CA A@EIJHE> KJE KHJDAH HA @=O)EI?D=H=?JAHE A@>OJDAANFHAIIE B= A IA > A BCA AIE L LA@E? FAJA?A= @@=O* >OJDAANFHAIIE BCA AIE L LA@E IF HK =JE Unidentified factors 6DAIA? JH A@= @K? JH A@B=?J HIIK?D=I? JHE>K JE B JDA GK= JEJO B HAIE@K= H4 ) E JDA E EJE= 4 ) FHAF=H=JE B K?JK=JE E JDAIJ=JA BJDA?K JKHA = EBAIJ JDA IA LAIE JDA @A 1 JDA HO JDAHAHA =E I=HAIE@K= JAH E JDAIJ=JEIJE?= @A )IJDAHAIE@KAEILAHOI = E JDAFHAIA JIJK@O JDEIE @E?=JAIJD=JK E@A JEBEA@B=?J HI@ JF =O= E F HJ= JH AE JDAANFAHE A JFHAIA JA@ comment reviews reports deposited research refereed research interactions information

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