Fig. S1

A

GrrA_Spl LISVLLVDDHELVRAGIRRILEDIKGIKVVGEAQCGEDAVKWCRGNTVDIVLMDMNMPGI 60

UvrY_Eco MINVLLVDDHELVRAGIRRILEDIKGIKVVGEASCGEDAVKWCRTNAVDVVLMDMSMPGI 60

ExpA_Pca MISVFLVDDHELVRAGIRRILDDIKGLKVVGEACCGEDAVKWCRSNDVDVVLMDMSMPGI 60

GacA_Pfl MIRVLVVDDHDLVRTGITRMLADIDGLQVVGQAESGEESLLKARELKPDVVLMDVKMPGI 60

:* *::****:***:** *:* **.*::***:* .**::: .* *:****:.****

GrrA_Spl GGLEATRKIVRYAPDVKVIMLTIHTENLLPAKVMQAGAAGYLSKGAAPQDVVNAIRSVQA 120

UvrY_Eco GGLEATRKIARSTADVKIIMLTVHTENPLPAKVMQAGAAGYLSKGAAPQEVVSAIRSVYS 120

ExpA_Pca GGLEATRKILRFMPDIKVIMLTIYTENPLPAKVMQAGAAGYVSKAAAPQEVICAIRAVHA 120

GacA_Pfl GGLEATRKLLRSHPDIKVVAVTVCEEDPFPTRLLQAGAAGYLTKGAGLNEMVQAIRLVFA 120

********: * .*:*:: :*: *: :*::::*******::*.*. :::: *** * :

HTH

GrrA_Spl GQRYIASDIAQQMALSQLEP-QTETPFSCLSERELQIMLMITKGKKVNEISEQLNLSPKT 179

UvrY_Eco GQRYIASDIAQQMALSQIEPEKTESPFASLSERELQIMLMITKGQKVNEISEQLNLSPKT 180

ExpA_Pca GKRYIASDIAQQMALSQLEP-QTDAPLECLSERELQIMLMITKGQKVTEISDQLNLSPKT 179

GacA_Pfl GQRYISPQIAQQLVFKSFQP-SSDSPFDALSEREIQIALMIVGCQKVQIISDKLCLSPKT 179

*:***:.:****:.:..::* .:::*: .*****:** ***. :** **::* *****

GrrA_Spl VNSYRYRMFSKLNISGDVELTHLAIRHGLFNAETLLSSE 218

UvrY_Eco VNSYRYRMFSKLNIHGDVELTHLAIRHGLCNAETLSSQ- 218

ExpA_Pca VNSYRYRMFSKLNINGDVELTHLAIKHGLFTAETLLSSE 218

GacA_Pfl VNTYRYRIFEKLSISSDVELTLLAVRHGMVDASL----- 213

**:****:*.**.* .***** **::**: *.

B

GrrS_Spl -MTKYSLRARMMILILAPTLLIGLLLSTFFVVHRYNELQEQLVDAGASIIEPLAVASEYG 59

ExpS_Pca -MTKYSLRARMLILILAPTLMIGLLLSSFFVIHRYNQLQTQLADSGTRIIEPLATISAYA 59

BarA_Eco -MTNYSLRARMMILILAPTVLIGLLLSIFFVVHRYNDLQRQLEDAGASIIEPLAVSTEYG 59

GacS_Pfl MLKKLGIKGRVLLLTLLPTSLMALVLGGYFTWMQLSDLQTQLLQRGEMIAEQLASLVAPA 60

:.: .::.*:::* * ** ::.*:*. :*. : .:** ** : * * * ** .

GrrS_Spl MTFRSRESVRQLVSLLHRRHSDIVRSITVFDAQNNLFVTSNYHHNFAQLQLPKGVPMPTD 119

ExpS_Pca ITHRQMDTIPTLINTLHRRHSAIIRTISVFDARNQLLATTNVRNNVTLQPLSSDALSYNK 119

BarA_Eco MSLQNRESIGQLISVLHRRHSDIVRAISVYDENNRLFVTSNFHLDPSSMQLGSNVPFPRQ 119

GacS_Pfl MGNHNTQMLERIATQALEQQD--VRAVSLLAPDRSLLAHAG-----PSMLNPPPAGNSSH 113

: :. : : : . .::. :*:::: . *:. :. . . .

GrrS_Spl LMLTRRGNSLILRTPILSESQYPDQN-ATDPVPQDNNLGYVAIELDLQSVRLRQYKEVFI 178

ExpS_Pca LHLHHTNDALILHMPIVNDSEFMPGG-VQPGTATP--LGYLVIELDTSTIRLQQYQEIFI 176

BarA_Eco LTVTRDGDIMILRTPIISESYSPDESPSSDAKNSQNMLGYIALELDLKSVRLQQYKEIFI 179

GacS_Pfl MMQRSGSDATRYQLPVFGRHRNLAGDLIPDE--SDRLLGWVELDLSHSGMLLRGYRNLFA 171

: .: : *:.. . **:: ::*. . : *: *:::*

GrrS_Spl STLLLLLCMCIAILFAYRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGFMLGELHMLKNG 238

ExpS_Pca AALLLLLSLGAAVIFAYRLMRDVTTPIRNMVDTVDRIRRGQLDSRVEGYMLGELDMLKNG 236

BarA_Eco SSVMMLFCIGIALIFGWRLMRDVTGPIRNMVNTVDRIRRGQLDSRVEGFMLGELDMLKNG 239

GacS_Pfl SLLLIPAGLAVTALLAVRMGRTINNPLTQIKQAVAQLKDGNLETRLPPLGSQELDELASG 231

: ::: : : ::. *: * :. *: :: ::* ::: *:*::*: **. * .*

GrrS_Spl INSMAMSLTAYLEEMQQNIDQATSDLRETLEQMEIDNVELDLAKKRAQEAARIKSEFLAS 298

ExpS_Pca INSMAMSLTAYHEEMQQNIDQATYDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN 296

BarA_Eco INSMAMSLAAYHEEMQHNIDQATSDLRETLEQMEIQNVELDLAKKRAQEAARIKSEFLAN 299

GacS_Pfl INRMASTLQNAQEELQHSIDQATEDVRQNLETIEIQNIELDLARKEALEASRIKSEFLAN 291

** ** :* **:*:.***** *:*:.** :**:*:*****:*.* **:********.

GrrS_Spl MSHELRTPLNGVIGFTRQMLKTEMSVTQTDYLQTIERSANNLLTIINDVLDFSKLEAGKL 358

ExpS_Pca MSHELRTPLNGVIGFTRQTLKTSLNTTQTDYLLTIERSANNLLNIINDVLDFSKLEAGKL 356

BarA_Eco MSHELRTPLNGVIGFTRLTLKTELTPTQRDHLNTIERSANNLLAIINDVLDFSKLEAGKL 359

GacS_Pfl MSHEIRTPLNGILGFTHLLQKSELTPRQLDYLGTIEKSADSLLGIINEILDFSKIEAGKL 351

****:******::***: *:.:. * *:* ***:**:.** ***::*****:*****

GrrS_Spl VLEHIPFSLRETLDEVIVLLAPSAHEKGLELTLDVHNDVPEQVIGDSLRLQQIITNLLGN 418

ExpS_Pca VLEDIPFSLHSTLDDVVMLLAHTAHEKGLELTLSIQNDVPEQYVGDPLRIQQIITNLLGN 416

BarA_Eco ILESIPFPLRSTLDEVVTLLAHSSHDKGLELTLNIKSDVPDNVIGDPLRLQQIITNLVGN 419

GacS_Pfl VLDSIPFNLRDLLQDTLTILAPAAHAKQLELVSLVYRDTPLSLVGDPLRLKQILTNLVSN 411

:*: *** *:. *::.: :** ::* * ***. : *.* . :**.**::**:***:.*

GrrS_Spl AIKFTETGNIDIRVELRGQQDHQVELEVQIHDTGIGISERQQSQLFQAFRQADASISRRH 478

ExpS_Pca AIKFTEQGNIDIRVEKRRQEHHQVQLEVQICDTGIGIAELQQSQLFQAFRQADTSISRRH 476

BarA_Eco AIKFTENGNIDILVEKRALSNTKVQIEVQIRDTGIGIPERDQSRLFQAFRQADASISRRH 479

GacS_Pfl AIKFTREGTIVARAMLEEEHEDSVQLRISIQDTGIGLSNQDVRALFQAFSQADNSLSRQP 471

*****. *.* . . . .*::.:.* *****:.: : ***** *** *:**:

GrrS_Spl GGTGLGLVITQKLVKEMGGDICFHSQLNRGSTFWFHITLELHEGMLSLPSSLPDLRGKTL 538

ExpS_Pca GGTGLGLVITQRLVKEMGGDISFQSQLNKGSTFWFHITLPLNPHTVPIEPVYTMLQGKHL 536

BarA_Eco GGTGLGLVITQKLVNEMGGDISFHSQPNRGSTFWFHINLDLNPNIIIEGPSTQCLAGKRL 539

GacS_Pfl GGTGLGLVISKRLIEQMGGEIGVDSTPGEGSEFWISLNLPKTRDDAEDLP-GPPLLGRRV 530

*********:::*:::***:* ..* ..** **: :.* . * *: :

GrrS_Spl GYIEANPMAAEATLNMLSVTSLVITHSPTLAQLPKKNYDFLLVGVPIPFRDNLAQHESKL 598

ExpS_Pca AYVEYHPIAAQATLDILSQTPLMVSYSPTFEQLPEGEFDILLLAIPAQYRNTLLDYTPRL 596

BarA_Eco AYVEPNSAAAQCTLDILSETPLEVVYSPTFSALPPAHYDMMLLGIAVTFREPLTMQHERL 599

GacS_Pfl AVLENHELARQALQHQLEDCGLEVTPFNTLEALTNGITGVHQSEQAIDLAVLGITTNDMS 590

. :* : * :. . *. * : *: *. .. .

GrrS_Spl KAALNIAGRVILALPCQSQINAEQLKQQGAIGCLIKPITSNRLFPLLRMEPPQRLLAAP- 657

ExpS_Pca RDICRRAPCVILALPSLAQMDAEQLKAFGVHACLSKPLAASRLLPLLQNSTLFQLSFLPS 656

BarA_Eco AKAVSMTDFLMLALPCHAQVNAEKLKQDGIGACLLKPLTPTRLLPALTEFCHHKQNTLLP 659

GacS_Pfl PERLSQHIWDLEHLGCKVLVLCPTTEQTLFHLSVPNPHSQLQAKPACTRKLRRALSDLVT 650

: * . : . : .: :* : : *

GrrS_Spl ------ERKRLPLTVMAVDDNPANLKLIGTLLAEQVEKTLLCESGEEALALARDNV 707

ExpS_Pca DTTSHQGTVRHPARLPLSVMAVDDNPANLKLIGTLLEEQVDTIILCESGTEAIAQAKMHQ 716

BarA_Eco ------VTDESKLAMTVMAVDDNPANLKLIGALLEDMVQHVELCDSGHQAVERAKQMP 711

GacS_Pfl PRRARSEPEETLSSRAPRVLCVDDNPANLLLIQTLLEDMGAKVLAVDNGYAALNAIQTEP 710

. *:.******** ** :** : :.* *: :

GrrS_Spl LDLILMDIQMPNIDGIRTGELIRQLP---HHNSTPIVAVTAHAVSGEREHLLQAGMDDYL 764

ExpS_Pca IDIILMDIQMPGMDGICASELIRQIP---HHATTPIIAVTAQTMTGEREHLLSSGMDDYL 773

BarA_Eco FDLILMDIQMPDMDGIRACELIHQLP---HQQQTPVIAVTAHAMAGQKEKLLGAGMSDYL 768

GacS_Pfl FDLVMMDVQMPGMDGRKSTEAIRQWESERHGTPLPIVALTAHAMANEKRALLQSGMDDYL 770

:*:::**:***.:** : * *:* * *::*:**::::.::. ** :**.***

GrrS_Spl AKPIDEAMLTRVLARYYSGEPAA------EPLLSLDWPLALRQAANKEDLAR 810

ExpS_Pca AKPIDEQMLKSVLTRHARKDPLKRDRADMVGLLSEHDDSQLSLDWALAQQQAANKPELAR 833

BarA_Eco AKPIEEERLHNLLLRYKPGSGIS-SRVVTPEVNEIVVNPNATLDWQLALRQAAGKTDLAR 827

GacS_Pfl TKPISERQLAQVVLKWT-GLALRNQGPERASERPELGLELQVLDQDEGLRLAAGKADLAA 829

:***.* * :: : ** . : **.* :**

GrrS_Spl DLLQMLLDFLPQVSERVQAVLDGTPDDSILDLIHKLHGSC-YSGVPRLKQLCFYLEQQLR 869

ExpS_Pca DLLQMLLDFLPEVQRKIENVLNGQTDDDIIELVHKLHGSCSYSGVPRLKRICRYLEQQLR 893

BarA_Eco DMLQMLLDFLPEVRNKVEEQLVGENPEGLVDLIHKLHGSCGYSGVPRMKNLCQLIEQQLR 887

GacS_Pfl DMLAMLLASLDADREAIKAARAANDQNALIERVHRLHGATRYCGVPQLRAACQRSETLLK 889

*:* *** * . :: . : ::: :*:***: *.***::: * * *:

GrrS_Spl KGVGNEELEPEWLELLDEIELVSQAARSHLESDRPTTS 907

ExpS_Pca KGVHANDLEPEWLELLDEIDNVNNAAQPHINPRLP--- 928

BarA_Eco SGTKEEDLEPELLELLDEMDNVAREASKILG------918

GacS_Pfl Q--EDAKAFAALDELDHAIGRLAAEARTNA------917

. . . ** . : : *

C

RpoS_Spl MNQN-TLKVNELQDDADFDENATEAESFDEKALVEEEASESDLA-EELLSQG---VTQRV 55

RpoS_Eco MSQN-TLKVHDLNEDAEFDENG--VEVFDEKALVELEPSDNDLAEEELLSQG---ATQRV 54

RpoS_Pca MSQS-TLKVNELHEDTDFEENG--LDVFDDKALAEEDTNDNDSAEDELLSQG---VPQRV 54

RpoS_Pfl MALSKEVPEFDIDDEVLLMESSIDMDSMSNNEGATPPSVRTKSKSSASLKQHKYIDYTRA 60

* . : ::.::. : *.. : :.:: . . .. . *.* *.

[ 1 ] [

RpoS_Spl LDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVPSRRRMIESNLRLVVKIARRYSNRGL 115

RpoS_Eco LDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVASRRRMIESNLRLVVKIARRYGNRGL 114

RpoS_Pca LDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVPSRRRMIESNLRLVVKIARRYNNRGL 114

RpoS_Pfl LDATQLYLNEIGFSPLLSPEEEVHFARLSQSGDPAGRKRMIESNLRLVVKIARRYVNRGL 120

********.***:****:.****:*** : ** ..*:***************** ****

2 ] [

RpoS_Spl ALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKE 175

RpoS_Eco ALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKE 174

RpoS_Pca ALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKE 174

RpoS_Pfl SLLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHVVKE 180

:*******************************************************:***

3

RpoS_Spl LNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKAL 235

RpoS_Eco LNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKAL 234

RpoS_Pca LNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVNRMLALNERITSVDTPLGGDSEKAL 234

RpoS_Pfl LNVYLRAARELTQKLDHEPSPEEIANLLEKPVAEVKRMLGLNERVSSVDVSLGPDSDKTL 240

******:****::*******.****: *:*** :*.*** ****::***..** **:*:*

] [ 4

RpoS_Spl LDILADEKDNGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGRE 295

RpoS_Eco LDILADEKENGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGRE 294

RpoS_Pca LDILADEKDNGPEDTTQDNDMKQNIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGRE 294

RpoS_Pfl LDTLTDDRPTDPCELLQDDDLSQSIDQWLSELTEKQREVVVRRFGLRGHESSTLEDVGLE 300

** *:*:: ..* : **:*:.*.* :** **. *****:.***** *:*::****** *

]

RpoS_Spl IGLTRERVRQIQVEGLRRLREILQLQGLSIEALFRE 331

RpoS_Eco IGLTRERVRQIQVEGLRRLREILQTQGLNIEALFRE 330

RpoS_Pca IGLTRERVRQIQVEGLRRLREILQVQGLSIEELFRE 330

RpoS_Pfl IGLTRERVRQIQVEGLKRLREILEKNGLSSESLFQ- 335

****************:******: :**. * **:

Fig. S1.

A. Amino-acid sequence alignment of S. plymuthica IC1270 GacA protein deduced from the cloned grrA sequence, with ExpA from P. carotovora subsp. carotovora (Pca), UvrY from E. coli (Eco) and GacA from P. fluorescens CHA0 (Pfl). The conserved aspartate (D) and lysine (K) residues in the phosphorylation sites characteristic of highly conserved phosphoryl receiver N-terminal domains found in similar regulatory proteins are enclosed in rounded-off rectangles. Residues in the C-terminal region that are conserved in the FixJ family are circled. An amino-acid stretch corresponding to a helix-turn-helix (HTH) DNA-binding motif found in the C-terminal portion of FixJ class transcription activators is bolded. In this, as well as in the two following alignments (B and C): (*), identical residues in all sequences; (:), conserved replacement, and (.), semiconserved replacement.

B. Amino-acid sequence alignments of the GacS-like protein of S. plymuthica IC1270, ExpS of P. carotovora subsp. carotovora (Pca), BarA of E. coli (Eco) and GacS (= ApdA) of P. fluorescens Pf-5 (Pfl). The common features of the proteins are marked according to (9), using GacS protein as a reference. Underlined amino-acid stretches are hydrophobic regions. Boxed residues indicate conserved sensor kinase domains (18). A black triangle indicates a presumptive autophosphorylation site (H-box) located at His-294 Conserved aspartate (Asp-663, 664), the phosphorylated aspartate (Asp-717) and Lys-772 residues typical of receiver domain are circled. A conserved C-terminal domain is in bold. The C-terminal portion of the deduced grrS gene product contains an additional phosphoryl acceptor site at His-844 in Hpt output domain (18).

C. Predicted amino-acid sequence of RpoS of S. plymuthica IC1270 (Spl) aligned with the sequences of P. fluorescens (Pfl), E. coli (Eco) and S. typhimurium (Sty). Functional regions 1 to 4 highly conserved among σ70 family sigma factors (24), are shown in brackets. Bolded amino-acid sequences indicate a predicted helix-turn-helix motif according to (24, 37).