comparative genomics and evolution of genes encoding … · encoding bacterial (p)ppgpp...

Rel RelA and SpoT Proteins 585

Comparative Genomics and Evolution of GenesEncoding Bacterial (p)ppGpp SynthetasesHydrolases(the Rel RelA and SpoT Proteins)

For correspondenceEmail GerhardMittenhuberbiologieuni-greifswalddegmittenhuberpaulde Tel +49-3834-864218 Fax +49-3834-864202

J Mol Microbiol Biotechnol (2001) 3(4) 585-600

copy 2001 Horizon Scientific Press

JMMB Research Article

Gerhard Mittenhuber

Institut fuumlr Mikrobiologie und Molekularbiologie Ernst-Moritz-Arndt-Universitaumlt F-L Jahnstr 15 D-17487Greifswald Germany

Abstract

In the gram-negative model organism Escherichia colithe effector molecule of the stringent response(p)ppGpp is synthesized by two different enzymesRelA and SpoT whereas in the gram-positive modelorganism Bacillus subtilis only one enzyme named Relis responsible for this activity Rel and SpoT alsopossess (p)ppGpp hydrolase activity BLAST searcheswere used to identify orthologous genes in databasesThe construction and bootstrapping of phylogenetictrees allowed classification of these orthologs Fourgroups could be distinguished With the exception ofNeisseria and Bordetella (β subdivision) the RelA andSpoT groups are exclusively found in the γ subdivisionof proteobacteria Two Rel groups representing theactinobacterial and the BacillusClostridium groupwere also identified The SpoT proteins are related tothe gram positive Rel proteins RelA proteins carrysubstitutions in the HD domain (Aravind and Koonin1998 TIBS 23 469-472) responsible for ppGppdegradation A theory for the evolution of thespecialized paralogous relA and spoT genes ispresented After gene duplication of an ancestral rel-like gene the spoT and relA genes evolved from theduplicated genes The distribution pattern of theparalogous RelA and SpoT proteins supports a newmodel of linear bacterial evolution (Gupta 2000 FEMSMicrobiol Rev 24 367-402) This model postulates thatthe γ subdivision of proteobacteria represents the mostrecently evolved bacterial lineage However twoparalogous closely related genes of Porphyromonasgingivalis (Cytophaga-Flavobacterium-Bacteroidesphylum) encoding proteins with functions probablyidentical to the RelA and SpoT proteins do not fit inthis model Completely sequenced genomes of severalobligately parasitic organisms (Treponema pallidumChlamydia species Rickettsia prowazekii) and theobligate aphid symbiont Buchnera sp APS as well asarchaea do not contain rel-like genes but they are

present in the Arabidopsis genome In crosslinkingexperiments using different analogs of ppGpp ascrosslinking reagents and RNA polymerasepreparations of Escherichia coli binding of ppGpp todistinct regions at the C-terminus of the β subunit (theRpoB gene product) andor at the N-terminus of the βsubunit (the RpoC gene product) was observedpreviously RpoB and RpoC sequences of the specieswhich do not possess a rel like gene do not exhibitspecific insertions or deletions in the ppGpp bindingregions

Introduction

The stringent response is an adaptive global mechanismfor control of gene expression in bacterial cells subjectedto starvation for amino acids or carbon sources (for reviewsee Cashel 1996) Initiallly discovered forty years ago inthe gram-negative model organism Escherichia coli (Stentand Brenner 1961) this response is characterized byprofound changes in the transcriptome of starved cells (egcessation of rRNA biosynthesis and activation of genesencoding amino acid biosynthesis genes activation of thestationary sigma factor σS (Gentry et al 1993) andtranscription at σS-dependent promoters (Kvint et al 2000)The stringent response is mediated through the synthesisof the effector molecule (p)ppGpp (Cashel and Gallant1969)

In E coli two different proteins are involved in(p)ppGpp synthesis RelA is bound to ribosomes sensesthe amount of uncharged tRNArsquos and synthesizes (p)ppGppfollowing amino acid limitation (Haseltine and Block 1973)The SpoT is a cytosolic protein (Gentry and Cashel 1995)and functions as (p)ppGpp synthetase after carbon(Hernandez and Bremer 1991 Murray and Bremer 1996)and fatty acid (Seyfzadeh et al 1993) starvation SpoT isalso a (p)ppGpp hydrolase (Hernandez and Bremer 1991Murray and Bremer 1996) The relA and spoT genes of Ecoli are related resulting in the hypothesis that these genesevolved by gene duplication (Metzger et al 1989) Residual(p)ppGpp synthesis in a relA mutant is abolished in a relAspoT double mutant (Xiao et al 1991) relAspoT doublemutants show a complex phenotype (morphologicalalterations loss in the ability to grow on amino acid-freeminimal media) (Xiao et al 1991 Hernandez and Cashel1995) The relA and spoT genes are listed as paralogousin the COG (cluster of orthologous groups) database(Tatusov et al 1997 Tatusov et al 2000) as COG0317

Several gram-positive bacterial species (ie Bacillussubtilis (Wendrich and Marahiel 1997) Corynebacteriumglutamicum (Wehmeier et al 1998) Mycobacteriumtuberculosis (Avarbock et al 1999) Staphylococcus

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Further Reading

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aureus (Gentry et al 2000) Streptococcus equisimilis(Mechold et al 1996 Mechold and Malke 1997)Streptomyces coelicolor (Chakraburtty et al 1996Martinez-Costa et al 1996 1998) Streptomycesantibioticus (Hoyt and Jones 1999)) however possess onlyone relAspoT paralog which exhibits both (p)ppGppsynthetase and hydrolase activity In these differentspecies ppGpp fulfills a variety of regulatory functions InB subtilis mutant strains defective in the relAspoT geneexhibit also pleiotropic phenotypes (amino acidauxotrophies (Wendrich and Marahiel (1997) defects inthe expression of several genes controlled by thesporulation sigma factor σH and reduced sporulationefficiency (Eymann et al 2001)) A rel mutant ofMycobacterium tuberculosis displays slower aerobic growthrate and is less able to survive extended anaerobicincubation (Primm et al 2000) The relAspoT gene is even

essential in S aureus (Gentry et al 2000) ppGpp plays arole in antibiotic and pigment production (Martinez-Costaet al 1996 Chakraburtty and Bibb 1997 Hoyt and Jones1999 Hesketh et al 2001) and morphologicaldifferentiation (Chakraburtty and Bibb 1997) inStreptomyces species

In the genomes of the gram-negative speciesMyxococcus xanthus (Harris et al 1998) andPorphyromonas gingivalis (Sen et al 2000) both relA andspoT genes seem to be present Harris et al (1998) clonedand sequenced the relA gene of M xanthus anddemonstrated that its gene product is essential for fruitingbody formation The other paralogous gene is not yetcloned Sen et al (2000) performed a search of theldquounfinished microbial genomerdquo sequence database of Pgingivalis at TIGR identified two homologues ORFrsquos andshowed that ORF1 encodes RelA of P gingivalis The otherORF is not yet characterized experimentally

Table 2 RelA orthologs The sequence of the RelA protein of E coli was used as query sequence inthe BLAST search Individual proteins are sorted according to ascending E-values For designationsof species see Table 1 The asterisk marks sequence fragments which were not included in thealignments because of their shortness Designations of proteins SpoT Pfr SpoT of Phlomobacterfragariae (Foissac et al 2000) SpoT SpoT sequence from an unidentified bacterium (Foissac et al2000) CsrS Vsp CsrS of Vibrio sp (Oumlstling et al 1995)

Designation Length Accession number E-value

RelA Eco 744 J04039 00RelA Vch 738 Q9S3S3 11e-263RelA Vsp 744 P55133 67e-262RelA Hin 743 P44644 35e-256RelA Pae 747 AAG04323 36e-183RelA Bha 728 BAB04961 11e-140RelA Bsu 734 U86377 24e-138RelA Xfa (XF1316) 718 AE003964 12e-136RelA Nme 769 CAB85211 76e-135Rel Ssp 760 P74007 8e-131Rel Sau 736 O32419 19e-129Rel Sco 847 X92520 75e-128Rel Cgl 760 O87331 41e-127Rel Mtu 790 Q50638 60e-126Rel Mle 787 Q49640 33e-125Rel Mxa 757 O52177 42e-125Rel Seq 739 Q54089 88e-125Rel San 841 O85709 23e-124Rel Aae 696 O67012 12e-123Rel Dra (DR1838) 787 Q9RTC7 26e-111SpoT Vch (VC2710) 705 AAF95850 3e-108SpoT Eco 702 P17580 17e-101SpoT Pae 701 AAG08723 17e-101SpoT Nme 725 CAB85138 23e-94SpoT Hin 677 P43811 87e-94Rel Tma (TM0729) 751 Q9WZI8 54e-93Rel Bja 779 Q9RH69 72e-84Rel Bbu 667 O51216 11e-80SpoT Xfa (XF0352) 735 AE003887 87e-77Rel Cje 731 AL139077 59e-74Rel Sci 749 O34098 62e-69RshA Sco (SC4H215) 725 O69970 86e-67Rel HpyJ99 776 Q9ZL68 76e-66F15I123 Ath 715 Q9SYH1 33e-59RSH2 Ath 710 AAF37282 34e-52SpoT Pfr 388 (fragment) AAG00076 30e-45T2H39 Ath 615 O81418 21e-40RSH1 Ath 883 AAF37281 24e-38Rel Uur 718 AE002125 33e-37Rel Mpn 733 P75386 71e-32Rel Mge 720 P47520 20e-29SpoT 283 (fragment) AAF73865 13e-27CsrS Vsp 119 (fragment) Q56730 1e-20

588 Mittenhuber

RelASpoT homologs have also been discovered inthe model plant Arabidopsis thaliana (van der Biezen etal 2000) These authors used the yeast two-hybrid assayin order to identify proteins that interact with RPP5 ThisArabidopsis protein which possesses a protein-proteininteraction module called ldquoNB-ARCrdquo (van der Biezen andJones 1998 Aravind et al 1999) is part of a signaltransduction cascade involved in sensing the fungalpathogen Peronospora parasitica (Noel et al 1999) Inthis two-hybrid assay the RSH1 (RelASpoT homolog)protein was identified a predicted plasma membrane-anchored cytoplasmic molecule with significant homologyto RelASpoT In a BLAST search two other unlinked rel-like genes were detected in the Arabidopsis genome theRSH2 and RSH3 genes (van der Biezen et al 2000)Functional complementation of appropriate E coli and Scoelicolor mutants by the RSH1 gene was also achievedin this study

Very recently the cloning and characterization of agene encoding a relAspoT homolog in Bacillusstearothermophilus was reported (Wendrich et al 2000)The authors include a phylogenetic tree of 32 proteinshomologous to RelASpoT of B subtilis van der Biezen etal (2000) also present a phylogenetic tree The RelA andSpoT proteins of E coli and closely related bacteria formdistinct clusters A cluster of actinobacterial RelASpoTsequences and a cluster of sequences representing theBacillusClostridium group can be observed Wendrich et

al (2000) also propose a nomenclature for enzymesinvolved in (p)ppGpp metabolism (p)ppGpp synthetase I(represented by RelA of E coli) (p)ppGpp synthetase II(represented by SpoT of E coli) and (p)ppGpp synthetaseIII (represented by synthetases of gram-positive origin thename Rel is proposed for this type of enzyme) Thisnomenclature is adopted in this paper althoughbiochemical evidence has not yet been reported for severalof the proteins named ldquoRelrdquo

However Wendrich et al (2000) and van der Biezenet al (2000) do not address the interesting question ofevolutionary relationships between these three classes ofenzymes to some detail In this paper these relationshipsare investigated using a larger number of RelA SpoT andRel sequences for construction of phylogenetic trees (fora list of species analyzed see Table 1) and by performingstatistical tests of tree topologies (ie bootstrapping(Felsenstein 1985)) During these studies we noted thatgenes encoding Rel-like proteins are absent in completelysequenced genomes of bacteria adopted to intracellularlifestyles Therefore we also investigated whether thetarget regions for binding of (p)ppGpp to the β and βsubunits of RNA polymerase (RNAP) in these speciesexhibit insertions or deletions (indels)

Results

Four General Groups of ppGpp SynthetaseHydrolaseProteinsOur initial phylogenetic analysis of the dataset of Wendrichet al (2000) showed a different tree topology than the onereported by the authors (data not shown) The reliability ofthe branching pattern of these tree data was then testedusing bootstrapping (Felsenstein 1985) Bootstrappinginvolves a randomization process in a multiple sequencealignment where all residues in a given column of thealignment are preserved in that column Pseudosamplesare created by random selection of sites from the data setwith replacement This is repeated a large number of times(eg 1000) and a phylogenetic tree is derived for eachpseudosample The percentage of pseudosamples whichsupports a unique clustering topology provides an estimateof the support for the pattern examined Two shortsequence fragments (ie CsrS from Vibrio sp (GenBankaccession number AAA85717 length 119 amino acidsOumlstling et al 1995) and a fragment derived from a gene ofPseudomonas denitrificans (GenBank acc No P29941length 175 aa Crouzet et al 1991) which were includedin the dataset of Wendrich et al (2000) were excluded fromthe dataset used for creation of the alignment becausebootstrapping of a dataset containing short sequencefragments is not informative A phylogenetic tree wasconstructed by the neighbor-joining (NJ) method (Saitouand Nei 1987) on the basis of the Poisson-corrected aminoacid distance (daa) Bootstrapping was performed with 1000replicates (Figure 1) This tree is mainly multifurcating (orldquocondensedrdquo) because many of the interior branchesexhibit a bootstrap value lower than 50 Only branchesseparating representatives of closely related species aswell as the RelA and SpoT proteins are supported by highbootstrap values

Figure 1 Rectangular cladogram of 30 RelA SpoT and Rel proteinsNumbers on the branches are percentages of 1000 bootstrap samplesthat support the branch only values gt50 are shown Bootstrapping wasperformed by the neighbor-joining (NJ) method (Saitou and Nei 1987) onthe basis of the Poisson-corrected amino acid distance (daa) The sequenceF15I1-23 of Arabidopsis was defined as outgroup


We assumed that a larger set of sequences used asinput file for the alignment might result in a more informativetree Therefore BLAST searches using the RelA (Table 2)and SpoT (Table 3) sequences of E coli and the Relsequence of B subtilis (Table 4) as query sequences wereperformed The Rel sequence of B subtilis was also usedto search the ldquounfinished microbial genomesrdquo database atTIGR (see Table 5) A dataset comprising 80 relatedsequences was obtained in this way An alignment of thesesequences was performed and an unrooted tree wasobtained (Figure 2) Several designations have beenreplaced by numbers in this tree A clear separation of theRelA and SpoT families on one hand and of two Rel familiesof gram-positive origin (BacillusClostridium group and aactinobacterial group) can be observed Bootstrapping ofthis dataset also resulted in a largely condensed tree withinternally supported branches (Figure 3) Interestingly as

also indicated by Wendrich et al (2000) and as depictedin Figure 1 mycoplasmal Rel proteins form a clearoutgroup

It seemed likely that exclusion of certain sequencesfrom the dataset used to produce the alignment mightincrease the resolution of the condensed part of the treeIn a first attempt the outgroup sequences of Arabidopsis(RSH2 Ath F15I1-23 Ath) and of the Mycoplasma (Rel MpnRel Mge Rel Uur) group as well as the Arabidopsissequences T2H3-9 and RSH1 which are located in thecondensed part of the tree were removed from the dataset(total 73 sequences) subjected to the alignment Theresulting bootstrapped tree did not exhibit increasedresolution in the condensed part of the tree (data notshown) In a second attempt internal sequences of thecondensed region of the tree in Figure 3 (ie Rel Dra RelSsp Rel Det Rel Aae Rel Tma Rel Mxa Rel Gsu Rel

Table 3 SpoT orthologs The sequence of the SpoT protein of E coli was used as query sequencein the BLAST search Individual proteins are sorted according to ascending E-values For designationsof species see Table 1 For explanation of the asterisk see Figure 2 Designation of the proteinSpoT Pde SpoT of Pseudomonas denitrificans (Crouzet et al 1991)


SpoT Eco 702 P17580 00SpoT Vch (VC2710) 705 AAF95850 19e-280SpoT Pae 701 AAG08723 66e-207SpoT Hin 677 P43811 23e-204SpoT Pfr 388 (fragment) AAG00076 11e-165SpoT Xfa (XF0352) 735 AE003887 37e-165SpoT Nme 725 CAB85138 13e-164Rel Bha 728 BAB04961 58e-148Rel Bsu 734 U86377 25e-147Rel Seq 739 Q54089 42e-143Rel Ssp 760 P74007 38e-142Rel Sau 736 O32419 8e-140Rel Mxa 757 O52177 19e-138Rel Aae 696 O67012 16e-134Rel San 841 O85709 17e-133Rel Mtu 790 Q50638 2e-132Rel Cgl 760 O87331 23e-131Rel Mle 787 Q49640 16e-130Rel Sco 847 X92520| 38e-127Rel Dra (DR1838) 787 Q9RTC7 55e-126Rel Tma (TM0729) 751 Q9WZI8 13e-125SpoT 283 (fragment) AAF73865 91e-123Rel Bja 779 Q9RH69 47e-116RelA Pae 747 AAG04323 95e-112RelA Nme 769 CAB85211 1e-105RelA Hin 743 P44644 94e-103Rel Bbu 667 O51216 51e-102RelA Eco 744 J04039 52e-100RelA Vsp 744 P55133 76e-97RelA Xfa (XF1316) 718 AE003964 29e-94Rel Cje 731 AL139077 34e-91Rel Sci 749 O34098 16e-90SC4H215 Sco 725 O69970 1e-88RelA Vch (VC249-51) 738 Q9S3S3 25e-88Rel Hpy99 776 Q9ZL68 15e-85RSH1 Ath 883 AAF37281 13e-73F15I123 Ath 715 Q9SYH1 96e-72Rel Uur 718 AE002125 12e-67RSH2 Ath 710 AAF37282 15e-67T2H39 Ath 615 O81418 81e-62Rel Mge 720 P47520 41e-56Rel Mpn 733 P75386 6e-55CsrS Vsp 119 (fragment) Q56730 11e-45SpoT Pde 175 (fragment) P29941 24e-10

590 Mittenhuber

Figure 2 Radial phylogenetic tree of 80 RelA SpoT and Rel proteins Due to space limitations the designation of individual Rel proteins was replaced bynumbers 1 refers to Rel Sco 2 to Rel San 3 to Rel Mav 4 to Rel Mle 5 to Rel Mtu 6 to Rel Mbo and 7 to Rel Cdiph


Dvu) were removed from the dataset (total 65 sequences)used for production of the alignment However use thisdataset in the construction of a bootstrapped tree also didnot result in increased resolution of the consensed part onthe tree (data not shown) In a third attempt seven otheroutgroup sequences (ie Rel Cte Rel1 Pgi Rel2 Pgi RelBbu Rel Sci Rel Cje and Rel Hpy) were removed fromthe dataset The alignment of this dataset (58 sequences)finally resulted in improved resolution of the resultingbootstrapped tree (Figure 4)

A statistically insignificant branching (53) separatesthe RelA proteins from the SpoT proteins and two groupsof Rel proteins from gram-positive species A statisticallysignificant branching (99 ) separates the Rel proteins ofthe BacillusClostridium group from the SpoT proteins andthe actinobacterial Rel proteins The SpoT proteins areseparated from the actinobacterial Rel proteins by astatistically less significant branching (78) An unrooted

radial tree originating from this alignment is presented inFigure 5 It should be noted that on the other hand whenanother dataset comprising only the sequencesrepresenting the condensed part of the tree of Figure 2was used as input file for an alignment this procedure alsodid not increase the resolution in the condensed part ofthe resulting bootstrapped tree (data not shown)

Substitutions in the HD Domain of RelA ProteinsIt was recognized by Aravind and Koonin (1998) that theHD domain defines a new superfamily of metal-dependentphosphohydrolases Aravind and Koonin (1998) define fivemotifs within the HD domain Three of them (motifs I IIand V) contain highly conserved histidine or aspartateresidues Site-directed mutagenesis experiments of genesencoding cGMP-phosphodiesterases have shown thatmutations of the histidine residue in the HD signature inmotif II or of the aspartate residue in motif V resulted in the

Table 4 Rel orthologs The sequence of the Rel protein of B subtilis was used as query sequence in the BLAST searchIndividual proteins are sorted according to ascending E-values For designations of species see Table 1 For explanation of theasterisk see Figure 2

Designation Length Accession number E-Value

Rel Bsu 734 U86377 00Rel Bha 728 BAB04961 2e-278Rel Sau 736 O32419 12e-241Rel Seq 739 Q54089 99e-197Rel Ssp 760 P74007 75e-167Rel Mxa 757 O52177 75e-167Rel Sco 847 X92520 26e-164Rel San 841 O85709 31e-161Rel Mtu 790 Q50638 29e-158Rel Mle 787 Q49640 23e-156Rel Cgl 760 O87331 24e-154Rel Aae 760 O67012 95e-148SpoT Eco 702 P17580 3e-140SpoT Pae 701 AAG08723 31e-138RelA Pae 747 AAG04323 35e-137Rel Tma (TM0729) 751 Q9WZI8 93e-137SpoT Vch (VC2710) 705 AAF95850 45e-135RelA Eco 744 J04039 68e-134RelA Vch 738 Q9S3S3 18e-133RelA Vsp 744 P55133 11e-131RelA Hin 743 P44644 57e-130SpoT Nme 725 CAB85138 37e-129SpoT Hin 677 P43811 22e-122RelA Nme 769 CAB85211 13e-121SpoT Xfa (XF0352) 735 AE003887 17e-120Rel Bbu 667 O51216 12e-104Rel Sci 749 O34098 14e-103RelA Xfa (XF1316) 718 AE003964 6e-103Rel Bja 779 Q9RH69 68e-102Rel Cje 731 AL139077 48e-93Rel Dra (DR1838) 787 Q9RTC7 27e-91RshA Sco (SC4H215) 725 O69970 98e-88Rel Hpy 776 Q9ZL68 13e-82Rel Uur 718 AE002125 28e-78T2H39 Ath 615 AAC28176 19e-71Rel Mge 720 P47520 53e-69Rel Mpn 733 P75386 23e-68F15I123 Ath (identical to RSH3) 715 AAD25787 12e-67RSH3 712 AAF37283 e-67SpoT Pfr 388 (fragment) AAG00076 33e-65RSH1 Ath 883 AAF37281 8e-64RSH2 Ath 710 AAF37282 11e-62SpoT 283 (fragment) AAF73865 39e-43CsrS Vsp 119 (fragment) Q56730 13e-25SpoT Pde 175 (fragment) P29941 18e-8

592 Mittenhuber

most severe effect on the catalytic activity (Turko et al1996) Aravind and Koonin (1998) showed that the RelAand SpoT proteins are also members of this superfamilyBut the RelA protein of E coli contains substitutions in thepredicted catalytic residues of this domain Aravind andKoonin (1998) suggest that the HD domain of RelA isinactivated but still retains its native structure An inactivephosphohydrolase domain helps to explain why RelAproteins are not able to degrade ppGpp On the other handthe N-terminus of SpoT encompassing the HD domain issuffient for ppGpp hydrolase activity (Gentry and Cashel1996) An alignment of the N-termini of the SpoT Rel andRelA proteins is presented in Supplementary Material (httpjmmbnetsupplementary) This alignment shows theregion around conserved motifs I and II and the regionaround conserved motif V Importantly all the proteins

identified in this study as putative RelA proteins carrysubstitutions in all conserved regions whereas Rel andSpoT proteins and the Arabidopsis paralogs possess afunctional HD domain (httpjmmbnetsupplementary)Interestingly in most substitutions of the HD signaturesequence insertion of a proline residue took place anamino acid known to influence protein folding (eg Eylesand Gierasch 2000) Nevertheless the SpoT Rel and RelAproteins share some overall similiarity in this region Thisanalysis also suggests that the protein designated RelA ofM xanthus by Harris et al (1998) is in fact a Rel or SpoTprotein

Comparison of Putative ppGpp Binding SitesDuring this analysis we noted that genes encoding(p)ppGpp synthetaseshydrolases are absent in thecompletely sequenced genomes of the obligate parasitesChlamydia pneumoniae and C trachomatis (Kalman et al1999) Rickettsia prowazekii (Andersson et al 1998) andTreponema pallidum (Fraser et al 1998) Similarly thegenome of an intracellular symbiont of aphids Buchnerasp APS (Shigenobu et al 2000) does not contain a RelAor SpoT gene although this extremely adapted bacteriumis a close relative of E coli It seems likely that the relgenes were deleted during the adaptation to the intracellularenvironment and the establishment of specialized lifestylesin a process called ldquoreductive evolutionrdquo (Andersson andKurland 1998)

Genetic studies (Glass et al 1986) using strainscarrying amber mutations in rpo genes and biochemicalinvestigations (Reddy et al 1995 Chatterji et al 1998Toulokhonov et al 2001) using various ppGpp analogs ascrosslinking agents identified RNAP of E coli as target forppGpp Glass et al (1986) concluded that ppGpp binds tothe β subunit encoded by rpoB and also identified acommon motif in purine-nucleotide binding proteins(GXXXXGK la Cour et al 1985) in the amino acidsequence of RpoB at residues 880 to 886 By a combinationof a variety of methods (SDS-PAGE analysis of the[32P]N3ppGpp (azido-ppGpp)-bound enzyme trypticdigestion and Western blots) Chatterji et al (1998)identified a 45 kDa fragment of the β subunit as bindingsite for ppGpp This fragment is located at the C-terminusand consists of residues 802-1211 1216 or 1223 (Chatterjiet al 1998) Using the smaller ppGpp analog 6-thio-ppGpphowever the β subunit encoded by rpoC was very recentlyidentified as target for ppGpp (Toulokhonov et al 2001)The binding site for ppGpp was determined at the N-terminus (between residues 29-102) of the β subunit Theauthors explain these conflicting results by the propertiesof the different crosslinking reagents and by the 3D modelof RNAP (Zhang et al 1999) This model shows that theN-terminus of β and the C-terminus of β are spatially closeand constitute an intertwined interface Toulokhonov et al(2001) postulate that binding of ppGpp to RNAP isallosteric that the binding site is modular and is locatedclose to the intersubunit interface of the N-terminal and C-terminal ends of β and β

In order to reveal the presence or absence of thedifferent identified ppGpp binding regions in the β and βsequences alignments of RpoB (Table 6 httpjmmbnetsupplementary) and RpoC (Table 7 httpjmmbnet

Figure 3 Rectangular cladogram of 80 RelA SpoT and Rel proteinsNumbers on the branches indicated are bootstrap values as described inthe legend to Figure 1


residues 1140 to 1260 of the RpoB alignment are presentin the proteobacterial and chlamydial sequences and againaround residues 1340 to 1420 in the proteobacterialmycobacterial and mycoplasmal sequences Mostinterestingly the RpoB and RpoC sequences of the specieswhich do not possess a rel like gene do not exhibit specific

Table 5 Rel orthologs The sequence of the Rel protein of B subtilis was used as query sequence in the BLAST search of the ldquounfinishedmicrobial genomesrdquo database at TIGR For designations of species see Table 1 Individual proteins are sorted according to ascending E-values

Designation Length Contig designation E-value

Rel Ban 727 gnl|TIGR_1392|banth_1489 00Rel Bst 707 (fragment) gnl|UOKNOR_1422|bstear_Contig408 00Rel Efa 737 gnl|TIGR|gef_10492 00Rel Cdi 735 gnl|Sanger_1496|cdifficile_Contig876 00Rel Spn 740 gnl|TIGR|Spneumoniae_3836 00Rel Smu 740 gnl|UOKNOR_1309|Smutans_Contig98 00Rel Spy 739 gnl|OUACGT_1315|Spyogenes_Contig1 00Rel Cac 740 gnl|GTC|Caceto_gnl 00Rel Sequ 719 (fragment) gnl|Sanger_1336|sequi_Contig474 00Rel Gsu 716 gnl|TIGR_35554|gsulf_57 00Rel Det 728 gnl|TIGR_61435|deth_1541 00Rel Mbo 738 gnl|Sanger_1765|mbovis_Contig403 e-171Rel Cdiph 759 gnl|Sanger_1717|cdiph_Contig2 e-166Rel Dvu 717 gnl|TIGR_881|dvulg_159 e-166Rel Mav 647 gnl|TIGR|Mavium_223 e-162SpoT Tfe 759 gnl|TIGR|t_ferrooxidans_6160 e-149SpoT Ype 707 (fragment) gnl|Sanger_632|Ypesits_Contig1008 e-148SpoT Sty 709 (fragment) gnl|Sanger_601|Styphi_CT18 e-148RelA Sty 744 gnl|Sanger_601|Styphi_CT18 e-142RelA Ype 744 gnl|Sanger_632|Ypesits_Contig854 e-146RelA Ppu 746 gnl|TIGR|pputida_10724 e-145Rel Cte 731 gnl|TIGR|Ctepidum_3499 e-143SpoT Spu 712 (fragment) gnl|TIGR_24|sputre_6408 e-142RelA Spu 735 gnl|TIGR_24|sputre_6426 e-142RelA Lpn 734 gnl|CUCGC_446|lpneumo_MF18110397 e-141RelA Pmu 739 gnl|CBCUMN_747|Pmultocida e-140SpoT Pmu 711 (fragment) gnl|CBCUMN_747|Pmultocida e-139SpoT Aac 712 (fragment) gnl|OUACGT_714|Aactin_Contig253 e-139SpoT Bbr 759 gnl|Sanger_518|bbronchi_Contig2526 e-140SpoT Bpe 759 gnl|Sanger_520|Bpertussis_Contig236 e-140RelA Aac 743 gnl|OUACGT_714|Aactin_Contig233 e-136SpoT Ngo 718 gnl|OUACGT_485|Ngon_Contig1 e-134RelA Hdu 735 gnl|HTSC_730|ducreyi e-133RelA Ngo 737 gnl|OUACGT_485|Ngon_Contig1 e-129RelA Bpe 737 gnl|Sanger_520|Bpertussis_Contig301 e-120SpoT Hdu 569 (fragment) gnl|HTSC_730|ducreyi e-111Rel1 Pgi 762 gnl|TIGR|Pgingivalis_GPGcon e-114Rel Ccr 743 gnl|TIGR|Ccrescentus_12574 e-113SpoT Lpn 530 (fragment) gnl|CUCGC_446|lpneumo_MF91102397 e-94Rel2 Pgi 746 gnl|TIGR|Pgingivalis_GPGcon e-81

Table 6 RpoB sequences compared in this study For designations ofspecies see Table 1

Designation Length Accession number

RpoB Eco 1342 RNEBCRpoB Bsu 1193 P37870RpoB Tpa 1178 O83269RpoB Bsp 1342 BAB12761RpoB Rpr 1374 O52271RpoB Ctr 1252 O84317RpoB Cpn 1252 BAA98291RpoB Hin 1343 P43738RpoB Sau 1182 P47768RpoB Mpn 1391 P78013RpoB Bbu 1155 AAB91501RpoB Mtu 1172 CAB09390

Table 7 RpoC sequences compared in this study


RpoC Eco 1407 RNECCRpoC Bsu 1199 P37871RpoC Sau 1057 P47770RpoC Mtu 1316 P47769RpoC Bsp 1407 BAB12760RpoC Hin 1415 P43739RpoC Rpr 1372 Q9ZE20RpoC Ctr 1396 O84316RpoC Cpn 1393 Q9Z999RpoC Tpa 1416 O83270RpoC Bbu 1377 O51349RpoC Mpn 1290 P75271

supplementary) amino acid sequences were performedOur Supplementary Material (httpjmmbnetsupplementary) shows that the common motif in purinenucleotide-binding proteins described above is present alsoin RpoB sequences of obligately parasitic bacteria and inthe symbiont Buchnera sp APS Large insertions around

594 Mittenhuber

alterations This might indicate that the binding sites forppGpp are still present in the β and β subunits of thesespecies

Discussion

Absence of ppGpp in Archaea Presence of ppGpp inPlantsGenes encoding (p)ppGpp synthetases are absent in allcompletely sequenced genomes of Archaea Sinceeubacterial RNA polymerase is the target for the regulatoryaction of (p)ppGpp and since archaea possess atranscriptional apparatus similar to that of eukaryotesinstead (Langer et al 1995) this difference betweenbacteria and archae might explain this absence Starvationstudies in halobacteria demonstrated stringent control ofstable RNA biosynthesis but both growth rate control andstringent control are probably governed by mechanismsthat operate in the absence of ppGpp (Cimmino et al 1993Scoarughi et al 1995)

The finding that Arabidopsis possesses RelASpoThomologs (van der Biezen et al 2000) might indicate thatplants have retained some aspects of the bacterialtranscription apparatus Indeed in Arabidopsis proteinssimilar to bacterial sigma factors (σ70) have been identified

as products of genes specifically expressed in leafs anddestined for chloroplasts (Isono et al 1997) Alsochloroplast genomes encode subunits of bacterial-typeRNA polymerases (Sugiura et al 1998 Turmel et al 1999Allison 2000) Furthermore ppGpp was detected in thealga Chlamydomonas reinhardtii (Heizmann and Howell1978)

Syntheny ConsiderationsSynteny studies (comparison of the relative positions ofgenes in the genome of different organisms) might be asource for auxiliary information to identify orthologousgenes in genome sequencing projects (Huynen and Bork1998) In a systematic comparison using 256 operons ofE coli and 100 operons of B subtilis as query sequencesItoh et al (1999) tried to identify operons showing aconserved order of orthologous genes in 11 completegenome sequences These authors found that operonstructures are very rarely conserved The sequences ofthe relA and spoT operons of E coli (but not the sequenceof the corresponding rel gene of B subtilis) were also usedas query sequences This analysis showed that no genomecontains operons showing exactly the same organizationas the spoT and relA operons of E coli However due tothe complexity of the spoT operon the function of this

Figure 4 Rectangular cladogram of 58 RelA SpoT and Rel proteins Four different groups can be distinguished Numbers on the branches indicated arebootstrap values as described in the legend to Figure 1


Figure 5 Radial phylogenetic tree of 58 RelA SpoT and Rel proteins

operon was misclassified as ldquoDNARNA processingrdquo in thisstudy For this reason a short description of the relA andspoT operons of E coli and the rel gene region of B subtilisis given

In E coli the relA gene is the first gene in an operoncontaining also the genes encoding the MazEF (ma-zehebrew for ldquowhat is itrdquo) antitoxintoxin module (Aizenmanet al 1996) Two promoters of the mazEF genes are

negatively autoregulated by MazE and MazF andexpression of the module is positively regulated by FIS(Marianovsky et al 2001) In B subtilis and other gram-positive bacteria the putative mazEF locus is locatedupstream of the sigB operon (Mittenhuber 1999) encodingthe general stress response (Hecker and Voumllker 1998)sigma factor σB and its regulatory proteins (Wise and Price1995) A third gene in the relA operon mazG encodes a

596 Mittenhuber

protein with unknown function A BLAST search revealedthat similar genes are present in many bacterial genomes(data not shown) They are designated as similiar to ahypothetical 261 kDa protein named YBL1 (accessionnumber P33653) of Streptomyces cacaoi (Urabe andOgawara 1992)

The spoT gene of Escherichia coli is the third gene inan operon consisting of five genes The order is gmk-rpoZ-spoT-trmH-recG Gmk encodes guanylate kinase (Gentryet al 1993) rpoZ the Ω-subunit of RNAP (Gentry andBurgess 1989) trmH a tRNA modifying enzyme (tRNA(Gm18) 2-O-methyltransferase) (Persson et al 1997) andrecG a DNA helicase (Lloyd and Sharples 1991 Kalmanet al 1992) Interestingly the first three genes are linkedto guanosine metabolism whereas the trmH and recG geneproducts play a role in RNA and DNA processing Lloydand Sharples (1991) identified a putative promoter nearthe end of spoT indicating that trmH and recG might betranscribed as separate unit

The organization of the rel loci of B subtilis M lepraeM tuberculosis S equisimilis and S coelicolor is similar(Wendrich and Marahiel 1997 Avarbock et al 1999)Upstream of the rel gene the apt genes (encoding adeninephosphoryltransferase catalyzing the formation ofadenosine monophosphate from adenine andphosphoribosyl pyrophosphate in a salvage reaction) andgenes encoding components of the protein exportmachinery (secDF) are located in the same direction anddownstream of rel the cypH genes encoding cyclophilin(a peptidyl-prolyl cis-trans isomerase) are located in theopposite direction

Proposal for the Evolution of the Rel RelA and SpoTProteinsThe Mollicutes (Mycoplasma and relatives) which arenormally associated with the BacillusClostridium group ofgram-positive bacteria form a distinct outgroup in the treesThese organisms undergo faster rates of evolution andquite regularly form outgroups in phylogenetic trees oforthologous protein sequences (Eisen 1998)

With the exception of Neisseria and Bordetella speciesboth belonging to the β-proteobacteria two different genesencoding enzymes involved in (p)ppGpp synthesis anddegradation namely RelA and SpoT are only found in theszlig and γ subdivision of proteobacteria The SpoT genes ofthis group are related to genes encoding the actinobacterialgroup and the BacillusClostridium group of Rel proteins(Figures 4 and 5 see also the E-values in Table 3) Sincecomplete protein sequences were compared theseparation of the RelA proteins from the Rel and SpoTproteins is most probably due to the fact that the Rel andSpoT proteins possess ppGpp hydrolase activity whereasthe RelA proteins lack this activity It is also interesting tonote that the paralogous genes of individual species arenot closely related to each other Multiple parallel geneduplications in individual species as origin of the relA andspoT genes can therefore be excluded

Facilitated by the knowledge of the enzymatic activitiesof the Rel RelA and SpoT proteins and based on somelogical speculation a model for evolution of the RelA andSpoT proteins within the β and γ subdivision ofproteobacteria is proposed

(1) The common ancestor of the β- and γ-proteobacteriapossessed a rel gene of actinobacterial origin The spoTgene evolved from this gene under adaptation of the geneproduct to carbon and fatty acid starvation(2) Following gene duplication the additional copy of thisancestral gene is able to adopt to new functions In thiscase this adoption of new function was most probablyinactivation of the HD domain loss of (p)ppGpp hydrolyzingactivity and adaptation to amino acid starvation This copyevolved to the relA gene of β- and γ-proteobacteria

The Special Case of P gingivalisP gingivalis however represents an interesting exceptionfrom the scenario described in the previous paragraphThis organism possesses a relA and a spoT gene (Sen etal 2000 httpjmmbnetsupplementary) which arehowever not related to the proteobacterial relA and spoTgenes (Figure 3) Both paralogs which are named Rel1Pgi and Rel2 Pgi in this paper are closely related to eachother and to Rel of Chlorobium tepidum (Figure 3) In Pgingivalis the evolution of the paralogous rel1 (spoT-related) and rel2 (relA-related) occured independently ofthe proteobacterial relA and spoT genes At the moment itis impossible to decide whether this paralogy representsan example of convergent evolution Alternatively lateralgene transfer of short catalytically active domains of ppGppsynthetaseshydrolases might have been involved in theevolution of the rel1 and rel2 genes of P gingivalis In orderto solve this question more sequences encoding ppGppsynthetaseshydrolases from representatives of the CFB(Cytophaga-Flavobacterium-Bacteroides) phylum andgreen sulfur bacteria should be determined and compared(see also next paragraph) Alternatively a carefulphylogenetic analysis of the individual domains of the RelRelA and SpoT proteins (Aravind and Koonin 1998) whichis however beyond the scope of this paper might help tosolve this issue Such an analysis has been performed forthe separate domains of the conserved HSP70 (DnaK)family Striking differences in the relative rate of amino acidreplacement in different rates were observed and wereinterpreted as evidence for functional divergence (Hughes1993)

ppGpp SynthetasesHydrolases and BacterialEvolutionA drastically different view of bacterial evolution has beenrecently postulated by Gupta (2000a b) This hypothesisplaces the γ subdivision of proteobacteria at the end of aevolutionary linear scheme According to this theory the γsubdivision of proteobacteria constitutes the most recentlyevolved bacterial group Therefore specialized paralogousspoT and relA genes might be a relatively new invention ofbacterial evolution which might explain this limiteddistribution pattern among the β- and γ-proteobacteriaSimilarly the (maybe more efficient) vitamin B6 biosynthesispathway of E coli is mainly restricted to the γ subdivisionwhereas another widely conserved biochemicallyuncharacterized pathway is presumably operating in otherspecies (Mittenhuber 2001) Other unique features of theγ-proteobacteria were recently recognized by Margolin(2000) in his study on prokaryotic cell division Genesencoding the ZipA FtsL and FtsN proteins are only found


in genomes of completely sequenced γ-proteobacteriaVery recently Eisen (2000) noted that the currentlyavailable datasets of completely sequenced genomes arenot representative of evolutionary diversity It might bepredicted that the availability of completely sequencedgenomes of proteobacterial species belonging tosubdivisions other than β and γ might help to identify theprecursor of the proteobacterial relA and spoT genes

ppGpp is Not Present in Obligately IntracellularSpeciesThe species lacking a rel-like gene require living cells fortheir replication and growth and cannot be cultivated invitro The absence of rel-like genes in genomes of obligatelyparasitic organisms was also detected in a comparativegenome analysis of B burgdorferi and T pallidum(Subramanian et al 1999) R prowazekii possesses afew short pieces of a pseudogene which show strongsequence similarity to the relAspoT homologs (Anderssonet al 1998 Zomorodipour and Andersson 1999) whereasno rel-like gene is present in the C trachomatis genome(Stephens et al 1998) Interestingly some cultivableintracellular pathogens (eg B burgdorferi andMycoplasma species among others) possess rel-likegenes In most cases cultivation of bacteria implies thatthe inoculum is able to form colonies Investigations onvertical sections of E coli colonies show that the colony iscomposed of different layers of bacteria containing alsononviable bacteria (Shapiro 1994) It is very likely thatmany cells in a developed colony are starved for nutrientsand that the stringent response is switched on in thesecells It might be extremely interesting to investigatewhether a functional connection between in vitro cultivabilityof bacterial species and the absence of genes encoding(p)ppGpp synthetaseshydrolases in obligate parasites canbe established experimentally

Experimental ProceduresUsing the deduced protein sequences of the relA and spoTgenes of E coli and of the rel gene of B subtilis as querysequences BLAST searches (Altschul et al 1997) of thenon-redundant protein database nrdb95 (Holm and Sander1998) were performed at httpdoveembl-heidelbergdeBlast2 using the blastp program BLAST searches ofunfinished microbial genomes using the deduced proteinsequence of the rel gene of B subtilis as query wereperformed at httpwwwncbinlmnihgovMicrob_blastunfinishedgenomehtml using the program tblastn RawDNA sequences were translated using the programldquoTranslate toolrdquo at httpwwwexpasychtoolsdnahtmlThe COG database can be assessed at httpwwwncbinlmnihgovCOG Alignments were generatedusing CLUSTALW (Thompson et al 1994) at httpwwwebiacukclustalw Radial phylogenetic trees wereconstructed using the data from the alignments with thehelp of the program TreeView (httptaxonomyzoologyglaacukrodtreeviewhtml 996) and edited usingthe program Metafile Companion (httpwwwcompanionsoftwarecom) Bootstrapping of datasets wasperformed by the neigbor-joining (NJ) method on the basisof the Poisson-corrected amino acid distance (daa) (Saitouand Nei 1987 for a discussion of different statistical

methods and their applications see Hughes 1999 Nei andKumar 2000) using MEGA 20 (httpwwwmegasoftwarenet Kumar et al 2001)

Acknowledgements

This work was performed in the lab of Michael Heckerwhose support is gratefully acknowledged I thank MichaelHecker Ulrich Zuber and an anonymous referee forcomments on the manuscript Sequences of unfinishedmicrobial genomes were obtained at the website of ldquoTheInstitute of Genomic Researchrdquo (TIGR) at httpwwwtigrorg Sequencing of unfinished genomes is inprogress at several institutions funded by a variety oforganizations This listing includes ldquoname of organism(institution(s) source of funding)rdquo and can be also viewedat httpwwwtigrorgtdbmdbmdbinprogresshtml Aactinomycetemcomitans (University of Oklahoma NationalInstitute of Dental Research (NIDR)) B anthracis (TIGROffice of Naval Research (ONR)Department of Energy(DOE)National Institute of Allergy and Infectious Diseases(NIAD)) B halodurans (Japan Marine Science andTechnology Center) B stearothermophilus (University ofOklahoma National Science Foundation (NSF) Bbronchiseptica B pertussis C difficile N meningitidis Ypestis (Sanger Centre Beowulf Genomics) C crescentusC tepidum D ethenogenes D vulgaris S putrefaciensT ferrooxidans (TIGRDOE) C acetobutylicum (GenomeTherapeutics DOE) C diphteriae (Sanger CentreWorldHealth Organization (WHO)Public Health LaboratoryBeowulf Genomics) G sulfurreducens (TIGRUniversityof Massachusetts AmherstDOE) H ducreyi (NIAID) Lpneumophila (Columbia Genome Center NIAID) Mavium V cholerae (TIGRNIAID) M bovis (Sanger CentreInstitut PasteurVLA Weybridge MAFFBeowulfGenomics) M leprae (Sanger Centre The New YorkCommunity Trust) N gonorrhoeae (University ofOklahoma NIAID) P multocida (University of MinnesotaUS Department of Agriculture - National Reserach Initiative(USDA-NRI)Minnesota Turkey Growers Association) Pgingivalis (TIGRForsyth Dental Center NIDR) Paeruginosa (University of WashingtonPathoGenesisCystic Fibrosis FoundationPathoGenesis) P putida (TIGRGerman Consortium DOEBundesministerium fuumlr Bildungund Forschung (BMBF) S typhi (Sanger CentreWellcome Trust) S aureus (TIGR NIAIDMerck GenomeResearch Institute (MGRI)) S pneumoniae (TIGR TIGRNIAIDMGRI) S coelicolor (Sanger CentreJohn InnesCentre Biotechnology and Biological Sciences ResearchCouncil (BBSRC)Beowulf Genomics)

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gro

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Geo

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Prot

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sub

divi

sion

Geo

bact

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Gsu

Haem

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onel

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neum

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Neiss

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sub

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goNe

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Shew

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Xyle

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thom

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estis

Prot

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erob

acte

riace

aeYp

e








588 Mittenhuber





Results










590 Mittenhuber










592 Mittenhuber



















594 Mittenhuber


Discussion











596 Mittenhuber













Experimental ProceduresUsing the deduced protein sequences of the relA and spoTgenes of E coli and of the rel gene of B subtilis as querysequences BLAST searches (Altschul et al 1997) of thenon-redundant protein database nrdb95 (Holm and Sander1998) were performed at httpdoveembl-heidelbergdeBlast2 using the blastp program BLAST searches ofunfinished microbial genomes using the deduced proteinsequence of the rel gene of B subtilis as query wereperformed at httpwwwncbinlmnihgovMicrob_blastunfinishedgenomehtml using the program tblastn RawDNA sequences were translated using the programldquoTranslate toolrdquo at httpwwwexpasychtoolsdnahtmlThe COG database can be assessed at httpwwwncbinlmnihgovCOG Alignments were generatedusing CLUSTALW (Thompson et al 1994) at httpwwwebiacukclustalw Radial phylogenetic trees wereconstructed using the data from the alignments with thehelp of the program TreeView (httptaxonomyzoologyglaacukrodtreeviewhtml Page 1996) and edited usingthe program Metafile Companion (httpwwwcompanionsoftwarecom) Bootstrapping of datasets wasperformed by the neigbor-joining (NJ) method on the basisof the Poisson-corrected amino acid distance (daa) (Saitouand Nei 1987 for a discussion of different statistical


Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber































586 MittenhuberTa

ble

1 A

lpha

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eoba

cter

ia β

sub

divi

sion

Nei

sser

iace

aeN

goNe

isser

ia m

enin

gitid

isPr

oteo

bact

eria

β s

ubdi

visi

on N

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eria

ceae

Nm

ePa

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tocid

aPr

oteo

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eria

γ s

ubdi

visi

on P

aste

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lace

aePm

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onas

gin

giva

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terio

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as a

erug

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aPr

oteo

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eria

γ s

ubdi

visi

on P

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as g

roup

Pae

Pseu

dom

onas

put

ida

Prot

eoba

cter

ia γ

sub

divi

sion

Pse

udom

onas

gro

upPp

uSa

lmon

ella

typh

iPr

oteo

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eria

γ s

ubdi

visi

on E

nter

obac

teria

ceae

Sty

Shew

anel

la p

utre

facie

nsPr

oteo

bact

eria

γ s

ubdi

visi

on A

ltero

mon

adac

eae

Spu

Spiro

plas

ma

citri

Firm

icut

es B

acillu

sC

lost

ridiu

m g

roup

Mol

licut

es S

piro

plas

mat

acea

eSc

iSt

aphy

loco

ccus

aur

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Firm

icut

es B

acillu

sC

lost

ridiu

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illus

Stap

hylo

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Sau

Stre

ptoc

occu

s eq

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rmic

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Bac

illus

Clo

strid

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gro

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trept

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rept

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similis

Firm

icut

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ridiu

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roup

Stre

ptoc

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ceae

Sequ

Stre

ptoc

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rmic

utes

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illus

Clo

strid

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gro

up S

trept

ococ

cace

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rept

ococ

cus

pneu

mon

iae

Firm

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es B

acillu

sC

lost

ridiu

m g

roup

Stre

ptoc

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ceae

Spn

Stre

ptoc

occu

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rmic

utes

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strid

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gro

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trept

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cace

aeSp

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rept

omyc

es a

ntib

iotic

usFi

rmic

utes

Act

inob

acte

ria A

ctin

obac

terid

ae A

ctin

omyc

etal

es S

trept

omyc

inea

e S

trept

omyc

etac

eae

San

Stre

ptom

yces

coe

licol

orFi

rmic

utes

Act

inob

acte

ria A

ctin

obac

terid

ae A

ctin

omyc

etal

es S

trept

omyc

inea

e S

trept

omyc

etac

eae

Sco

Syne

choc

ystis

sp

PC

C68

03C

yano

bact

eria

Chr

ooco

ccal

esSs

pTh

erm

otog

a m

aritim

aTh

erm

otog

ales

Tma

Thio

bacil

lus

ferro

oxid

ans

Prot

eoba

cter

ia γ

sub

divi

sion

Tfe

Trep

onem

a pa

llidum

Spiro

chae

tale

s S

piro

chae

tace

aeTp

aUr

eapl

asm

a ur

ealyt

icum

Firm

icut

es B

acillu

sC

lost

ridiu

m g

roup

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licut

esU

urVi

brio

cho

lera

ePr

oteo

bact

eria

γ s

ubdi

visi

on V

ibrio

nace

aeVc

hVi

brio

sp

Prot

eoba

cter

ia γ

sub

divi

sion

Vib

riona

ceae

Vsp

Xyle

lla fa

stid

iosa

Prot

eoba

cter

ia γ

sub

divi

sion

Xan

thom

onas

gro

upXf

aYe

rsin

ia p

estis

Prot

eoba

cter

ia γ

sub

divi

sion

Ent

erob

acte

riace

aeYp

e








588 Mittenhuber





Results










590 Mittenhuber










592 Mittenhuber



















594 Mittenhuber


Discussion











596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber






































588 Mittenhuber





Results










590 Mittenhuber










592 Mittenhuber



















594 Mittenhuber


Discussion











596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber






































590 Mittenhuber










592 Mittenhuber



















594 Mittenhuber


Discussion











596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber







































592 Mittenhuber



















594 Mittenhuber


Discussion











596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber











































594 Mittenhuber


Discussion











596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber




































596 Mittenhuber















Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber




































Acknowledgements


References




598 Mittenhuber





































































600 Mittenhuber































598 Mittenhuber





































































600 Mittenhuber



































































600 Mittenhuber































comparative genomics and evolution of genes encoding … · encoding bacterial (p)ppgpp...

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