Identification of attenuation and antitermination regulation in prokaryotes

gb-2002-3-6-preprint0003 GBJ Deposited research article Identification of attenuation and antitermination regulation in prokaryotes Lathe C Warren III lathe@embl-heidelberg.de Suyama Mikita Bork Peer

EMBL, Meyerhofstr. 1, D69012 Heidelberg, Germany

Max Delbrück Center for Molecular Medicine, D13092 Berlin-Buch, Germany

These authors contributed equally

Genome Biology 1465-6906 2002 3 6 preprint0003.1 preprint0003.60 http://genomebiology.com/2002/3/6/preprint/0003 This is the first version of this article to be made available publicly, and no other version is available at present. The article was submitted to Genome Biology for peer review. 10.1186/gb-2002-3-6-preprint0003 23 4 2002 30 4 2002 2002 BioMed Central Ltd

Identification of attenuation and antitermination regulation in prokaryotes

The prediction of genes with attenuation or antitermination regulation is reported. The method is based on known characteristics of upstream sequences of regulated genes. We analyzed 26 complete genomes and are thus able to give a more complete picture of attenuation regulation in prokaryotes.

Abstract

Many operons of biochemical pathways in bacterial genomes are regulated by processes called attenuation and antitermination. Though the specific mechanism can be quite different, attenuation and antitermination in these operons have in common the termination of transcription by a RNA 'terminator' fold upstream of the first gene in the operon. In the past, detecting regulation by attenuation or antitermination has often been a long process of experimental trial and error, on a case by case basis. We report here the prediction of over 290 upstream regions of genes with attenuation or antitermination regulation structures in the completed genomes of Bacillis subtilis and Escherichia coli for which extensive experimental studies have been done on attenuation and antitermination regulation. These predictions are based on a computational method devised from characteristics of known terminator fold candidates and benchmark regions of entire genomes. We extend this methodology to 24 additional complete genomes and are thus able to give a more complete picture of attenuation and antitermination regulation in bacteria.

Bioinformatics Molecular biology Genetics Genome studies

Background

The control of gene expression can occur at many points in the transcription and translation of the genes of bacterial operons. Two mechanisms of operon regulation of great interest are "attenuation" and "antitermination" [1,2,3,4,5]. These mechanisms regulate the early termination of transcription of a wide variety of operons in diverse species. Classically, attenuation occurs when the transcribed RNA upstream of an operon has the ability to fold into two mutually-exclusive RNA-fold structures, one which is termed an antiterminator and the other a terminator. If the terminator hairpin loop is allowed to fold, transcription is ultimately halted. Alternatively, if the antiterminator structure folds, the terminator is precluded from folding and transcription of the operon proceeds. The mechanisms that alternate between these two RNA folds (terminators and antiterminators) are quite diverse. Regulation by antitermination (not to be confused with the alternative antiterminator fold of attenuation) can be differentiated from attenuation by the fact that alteration of the transcription complex (rather than alternate RNA structures) decreases the efficiency of downstream terminators. Though, in reality, the boundary between these two types of regulation is not distinct [3].

Attenuation and antitermination mechanisms have both been described in a wide variety of regulatory and biochemical pathways. These include operons involved in aminoacyl-tRNA biosynthesis, catabolic metabolism, amino-acid biosynthesis, ABC transport systems, ribosomal structural peptides and several others. They have been characterized in genomes as disparate as the low-GC gram-positive Bacillis subtilis and the proteobacteria Escherichia coli. The precise mechanisms that cause the attenuation or antitermination of these operons can be quite distinct. For example, the trp operons of E. coli [4,5] and B. subtilis [6,7], though both regulated by attenuation, are controlled by quite different mechanisms. Other operons, such as the structural ribosomal S10 operon of E. coli [8,9] are regulated by yet a different mechanism. Even between closely related species, the attenuation and antitermination, and upstream regulatory sequences can be entirely different.

Yet, one common and necessary feature of most experimentally described attenuation and antitermination mechanisms is an intrinsic terminator RNA fold structure [2,3]. The stem-loop structure of an intrinsic terminator has been well described [10] and the understanding of the mechanisms of termination has made great progress in recent years [11,12]. This structure is not only found at the location of 'standard' termination of transcription at the end of transcriptional units, but is also, by definition a part of attenuation and antitermination regulation. The major characteristics of this standard terminator structure is that it is relatively short, is energetically stable, has a G/C rich stem, contains a small terminal loop structure and, importantly, also contains a run of U residues on the 3' side of the stem-loop structure [10]. These characteristics of intrinsic terminators have been used in the past to predict terminator structures at the end of transcriptional units (operons) and thus assist in the prediction of transcriptional units in complete genomes [10,13] and on a limited basis for predicting regulatory attenuators [14]. Here we focus on using the characteristics and position of intrinsic terminators to predict and characterize attenuation and antitermination regulation in operons of B. subtilis and E. coli. These mechanisms of regulation are well described in these two genomes. We extend this characterization to an additional 24 genomes representative of the diversity of eubacteria and archeabacteria to give a broader picture of attenuation and antitermination regulation in prokaryotes and in a more automated and extensive manner than previously achieved.

Results

Characterization of attenuators in B. subtilis and E. coli

An extensive literature search for operons in B. subtilis regulated by attenuation or antitermination was conducted and 46 such operons were found. These range from the experimentally well described trp operon to those operons where terminator structures have been found and attenuation is expected though not well characterized experimentally [15,16] (for a full list see http://www.bork.embl-heidelberg.de/Docu/attenuation). These 46 known terminator structures were employed to determine common characteristics of B. subtilis attenuation terminators. Using these characteristics, we screened upstream regions of 3650 B. subtilis genes (using procedures described in Materials and Methods) for terminator folds. Forty-three of the original 46 known terminators found in the literature search were retained in this screening. An additional 1117 upstream folds that fit our criteria were also obtained. In addition, as a control, we used the same filtering and folding methodology on intergenic regions after the sequences were shuffled randomly (952 folds of randomly shuffled sequences were obtained after filtering).

The resulting folds of all intergenic regions and shuffled sequences obtained after filtering were plotted in terms of their stability and length (Figure 1). The known terminator folds lie in a cluster clearly separate and distinct from those folds of randomly shuffled sequences. Terminator folds are of a lesser free energy (ΔG) in relation to length than predicted folds of random sequences. A similar pattern of two easily separated clusters emerges when comparing known terminator structures with folded intragenic regions in which terminator are not expected to be found (data not shown).

Figure 1

Stability and length distributions of stem-loop structures in upstream sequence segments of B. subtilis

Stability and length distributions of stem-loop structures in upstream sequence segments of B. subtilis. The red line shows the largest variance (see Materials and Methods) derived from stem-loop structures in shuffled sequences. Light blue lines give the significance measurements based on standard deviation. The definition for each point together with the orientation of neighboring genes are shown in upper right panel.

Using principal component analysis, we determined the greatest variance of the randomly shuffled sequences. This can give us a measure (using standard deviation) of which folds are significantly different from folds of random sequences (see Materials and Methods). Of the 1160 folds, a total of 203 folds of intergenic regions obtained in our screen fall below the 2nd deviation line (Z ≤ -2) derived from the principal component. These are thus considered significantly different from random folds and possible terminations sites of attenuation or antitermination regulation. Forty-two of these are the known attenuation terminators folds (of the original 43 known folds maintained after filtering). Thus we are able to obtain 91.3% (42/46) of the known and experimentally characterized attenuation and antitermination sites using our filter and significance measure. Additionally, the filter and significance measure screens out over 97.7% (930 of 952) of the folds of random sequences. One hundred and sixty-one (203 total excluding 42 known) folds under the line (Z ≤ -2) are folds not yet analyzed experimentally and could be predicted to be attenuation terminator structures.

A detailed investigation found many of these predictions are strongly supported as a putative attenuation or antitermination sites by genomic context such as the presence of putative promoter sequences, upstream location of putative and known operons, etc. Two terminator structures upstream genes ydbJ and yqhI serve as detailed examples of how genomic context can inform and strongly support the predictions made in Table 1 (Figure 2). Gene ydbJ of B. subtilis is listed as hypothetical with homology to an ABC transporter gene (ATP-binding protein involved in copper transport). The gene immediately downstream, ydbK, has homology to membrane spanning permeases. Using STRING (a search tool for find recurring instances of neighboring genes [17]), orthologs of these two genes are also found in the same order in transcriptional units of 15 other distantly related genomes, suggesting the possibility these genes form an operon. These genes appear to be in a typical ABC transporter operon configuration and several ABC transporter operons are known to be regulated by attenuation in B. subtilis [15,16]. The ydbJ upstream region also has a putative promoter sequence and predicted folds using RNAfold (See Materials and Methods) of the entire upstream sequence suggest it can fold in complex possible antitermination folds (data not shown). Based on this context, we predict this is an ABC transporter operon regulated by attenuation. The second example, yqhI, is the first gene of a run of three genes all having homology to glycine biosynthesis genes in a putative transcriptional unit. This run of three genes also has orthologs found as neighbors in other genomes [17]. Many amino acid biosynthesis operons in B. subtilis are known to be regulated by attenuation [16], thus supporting this prediction.

Figure 2

Schematic drawing of the neighborhood and predicted structures for the B. subtilis genes ydbJ and yqhI

Schematic drawing of the neighborhood and predicted structures for the B. subtilis genes ydbJ and yqhI. Genes are signified by colored arrows and are in orientation of transcription in relation to orientation of reference gene (ydbJ or yqhI). Large blue stem-loop cartoons signify predicted terminator fold in attenuation, 't' is an annotated standard terminator fold. Intergenic regions are drawn to scale and bp lengths of these are given underneath figure.

Table 1

Predicted attenuators in the genome of B. subtilis

Z-score

Known

Upstream promoter^a

Gene^b

BS0929

-8.895

√

glycerol-3-phosphate dehydrogenase (glpD)

BS2825

-8.473

√

acetolactate synthase (acetohydroxy-acid synthase) (large subunit) (ilvB)

BS1310

-6.836

similar to chaperonin (ykkC)

BS0983

-6.733

√

hypothetical protein (yhaW)

BS3920

-6.468

√

PTS beta-glucoside-specific enzyme IIABC component (bglP)

BS1257

-6.332

RNA polymerase PBSX sigma factor-like (xpf)

BS2733

-6.238

√

alanyl-tRNA synthetase (alaS)

BS0013

-6.126

√

seryl-tRNA synthetase (serS)

BS2184

-5.942

√

hypothetical protein (yphP)

BS3966

-5.840

√

hypothetical protein (iolD)

BS2749

-5.839

√

histidyl-tRNA synthetase (hisS)

BS2520

-5.815

√

glycyl-tRNA synthetase (alpha subunit) (glyQ)

BS3396

-5.767

√

similar to amino acid permease (yvbW)

BS0215

-5.526

√

glycerol-3-phosphate permease (glpT)

BS2255

-5.344

hypothetical protein (ypiA)

BS2600

-5.342

similar to phage-related protein (yqbK)

BS1544

-5.239

√

isoleucyl-tRNA synthetase (ileS)

BS3710

-5.196

√

CTP synthetase (ctrA)

BS3895

-5.191

√

similar to pyrimidine nucleoside transport protein (yxjA)

BS3798

-5.164

√

PTS sucrose-specific enzyme IIBC component (sacP)

BS2614

-5.152

√

hypothetical protein (yqaQ)

BS2484

-5.148

√

similar to 5-formyltetrahydrofolate cyclo-ligase (yqgN)

BS1319

-5.124

√

cobalamin-independent methionine synthase (metC)

BS2682

-4.990

√

hypothetical protein (yraL)

BS3750

-4.958

√

threonyl-tRNA synthetase (thrZ)

BS0183

-4.938

√

NADH dehydrogenase (subunit 5) (ndhF)

BS3440

-4.890

√

levansucrase (sacB)

BS2715

-4.841

√

similar to formate dehydrogenase (yrhE)

BS2204

-4.819

√

xanthine phosphoribosyltransferase (xpt)

BS2139

-4.756

√

similar to N-acetylmuramoyl-L-alanine amidase (yomC)

BS2803

-4.750

√

valyl-tRNA synthetase (valS)

BS0093

-4.747

√

serine acetyltransferase (cysE)

BS3877

-4.696

√

hypothetical protein (yxkD)

BS1357

-4.680

√

hypothetical protein (ykrT)

BS0178

-4.675

√

L-glutamine-D-fructose-6-phosphate amidotransferase (glmS)

BS1658

-4.644

√

DNA polymerase III (alpha subunit) (polC)

BS3894

-4.630

√

hypothetical protein (yxjB)

tRNA-Arg

-4.609

tRNA-Arg (trnJ-Arg)

BS0535

-4.505

similar to antibiotic resistance protein (ydfB)

BS3839

-4.472

√

tyrosyl-tRNA synthetase (tyrZ)

BS2961

-4.430

√

tyrosyl-tRNA synthetase (tyrS)

BS0441

-4.399

hypothetical protein (ydbB)

BS1143

-4.382

√

tryptophanyl-tRNA synthetase (trpS)

BS1737

-4.375

√

similar to ribonucleoprotein (ymaA)

BS0254

-4.309

√

hypothetical protein (yczA)

BS1971

-4.297

√

similar to adenosylmethionine-8-amino-7-oxononanoate aminotransferase (yodT)

BS0643

-4.279

√

phosphoribosylaminoimidazole carboxylase I (purE)

BS3026

-4.270

√

leucyl-tRNA synthetase (leuS)

BS2889

-4.249

√

threonyl-tRNA synthetase (thrS)

BS3690

-4.191

√

hypothetical protein (ywlC)

BS1188

-4.176

√

similar to cystathionine gamma-synthase (yjcI)

BS0521

-4.158

√

hypothetical protein (ydeI)

BS2452

-4.143

√

similar to aminomethyltransferase (yqhI)

BS3604

-4.128

√

hypothetical protein (ywrE)

BS1313

-4.086

√

gamma-glutamyl kinase (proB)

BS3269

-3.989

√

similar to ABC transporter (ATP-binding protein) (yusC)

BS0423

-3.974

√

hypothetical protein (ydaH)

BS2504

-3.896

√

similar to transcriptional regulator (Fur family) (yqfV)

BS3900

-3.884

√

endo-beta-1,3-1,4 glucanase (bglS)

BS0038

-3.883

√

methionyl-tRNA synthetase (metS)

BS2319

-3.879

√

hypothetical protein (ypuF)

BS1683

-3.845

similar to phage-related protein (ymfE)

BS1881

-3.805

√

phosphoenolpyruvate synthase (pps)

BS3513

-3.805

√

putative membrane protein (csbA)

BS0432

-3.796

hypothetical protein (ydaO)

BS3355

-3.795

hypothetical protein (yvaI)

BS2858

-3.784

√

phenylalanyl-tRNA synthetase (alpha subunit) (pheS)

BS3668

-3.780

√

hypothetical protein (ywmD)

BS0927

-3.776

√

glycerol uptake facilitator (glpF)

BS0281

-3.739

√

hypothetical protein (ycdC)

BS2306

-3.714

RNA polymerase ECF-type sigma factor (sigma-X) (sigX)

BS0143

-3.709

√

RNA polymerase (alpha subunit) (rpoA)

BS0449

-3.687

√

similar to ABC transporter (ATP-binding protein) (ydbJ)

BS1094

-3.668

√

hypothetical protein (yitC)

BS1370

-3.645

√

motility protein A (motA)

BS3785

-3.618

√

hypothetical protein (ywdL)

BS0104

-3.612

√

ribosomal protein L10 (BL5) (rplJ)

BS3313

-3.584

similar to iron-binding protein (yvrC)

BS0716

-3.572

√

hypothetical protein (yetH)

BS1889

-3.524

√

response regulator aspartate phosphatase (rapK)

BS0673

-3.492

hypothetical protein (yerQ)

BS0002

-3.480

DNA polymerase III (beta subunit) (dnaN)

BS0880

-3.473

√

transcriptional regulator (senS)

BS1548

-3.473

√

transcriptional attenuator and uracil phosphoribosyltransferase activity (minor) (pyrR)

BS1409

-3.467

√

hypothetical protein (ykuH)

BS2881

-3.464

initiation factor IF-3 (infC)

BS1651

-3.412

uridylate kinase (smbA)

BS3888

-3.412

√

hypothetical protein (yxjH)

BS3889

-3.412

√

hypothetical protein (yxjG)

BS0925

-3.407

√

similar to adenosylmethionine-8-amino-7-oxononanoate aminotransferase (yhxA)

BS1205

-3.351

hypothetical protein (yjdG)

BS3784

-3.335

hypothetical protein (spsA)

BS3093

-3.325

√

hypothetical protein (yuaJ)

BS1470

-3.265

√

hypothetical protein (yktD)

BS1549

-3.265

√

uracil permease (pyrP)

BS2841

-3.262

√

aspartokinase II (lysC)

BS2903

-3.222

DNA polymerase I (polA)

BS3446

-3.202

√

hypothetical protein (yvdQ)

BS2984

-3.202

hypothetical protein (ytmQ)

BS1920

-3.201

√

similar to ATP-dependent DNA helicase (yocI)

BS3343

-3.192

√

hypothetical protein (yvgV)

BS3731

-3.192

√

hypothetical protein (ywiA)

BS3278

-3.188

similar to 3-hydroxyacyl-CoA dehydrogenase (yusL)

BS3412

-3.180

transcriptional regulator (LacI family) (lacR)

BS2278

-3.156

hypothetical protein (yphE)

BS3914

-3.141

√

hypothetical protein (yxiF)

BS1550

-3.120

√

aspartate carbamoyltransferase (pyrB)

BS0878

-3.120

√

thiamin biosynthesis protein (thiA)

BS2775

-3.111

√

similar to sodium/proton-dependent alanine carrier protein (yrbD)

BS1673

-3.111

dipicolinate synthase subunit A (spoVFA)

BS2569

-3.086

√

site-specific DNA recombinase (spoIVCA)

BS2713

-3.075

√

similar to formate dehydrogenase (yrhG)

BS2539

-3.075

heat-shock protein (dnaJ)

BS3719

-3.068

similar to cardiolipin synthetase (ywiE)

BS2711

-3.061

√

similar to methyltransferase (yrhH)

BS1166

-3.059

√

transcriptional regulator (tenA)

BS1446

-3.057

aminopeptidase (ampS)

BS0503

-3.033

√

hypothetical protein (yddM)

BS1455

-3.014

√

hypothetical protein (ykzG)

BS3164

-2.969

√

transcriptional regulator (comQ)

BS1360

-2.966

√

similar to ribulose-bisphosphate carboxylase (ykrW)

BS1536

-2.942

√

similar to acetylornithine deacetylase (ylmB)

BS3133

-2.940

√

similar to polyribonucleotide nucleotidyltransferase (yugI)

BS3635

-2.930

√

flagellar basal-body rod protein (flhO)

BS1659

-2.908

√

hypothetical protein (ylxS)

BS2216

-2.896

hypothetical protein (ypsA)

BS0115

-2.894

√

ribosomal protein S10 (BS13) (rpsJ)

BS0637

-2.878

√

hypothetical protein (yebB)

BS2627

-2.867

similar to transcriptional regulator (phage-related) (Xre family) (yqaE)

BS0745

-2.856

√

similar to quinone oxidoreductase (yfmJ)

BS1785

-2.852

transcriptional regulator (lexA)

BS1737

-2.805

√

similar to ribonucleoprotein (ymaA)

BS1525

-2.800

√

cell-division initiation protein (divIB)

BS2138

-2.782

hypothetical protein (yomD)

BS2162

-2.776

√

hypothetical protein (yokC)

BS1335

-2.740

√

similar to transcriptional regulator (MarR family) (ykoM)

BS0241

-2.658

√

similar to histidine permease (ybgF)

BS2715

-2.649

√

similar to formate dehydrogenase (yrhE)

BS1855

-2.649

√

similar to phosphoglycerate dehydrogenase (yoaD)

BS2986

-2.643

√

hypothetical protein (ytmP)

BS1321

-2.642

hypothetical protein (ispU)

BS0386

-2.640

√

similar to transcriptional regulator (TetR/AcrR family) (ycnC)

BS2425

-2.616

similar to exodeoxyribonuclease VII (large subunit) (yqiB)

BS0057

-2.589

similar to amino acid transporter (yabM)

BS3957

-2.586

similar to ABC transporter (ATP-binding protein) (yxdL)

BS1003

-2.585

√

Hit-like protein (hit)

BS3350

-2.580

hypothetical protein (yvaC)

BS1659

-2.573

√

hypothetical protein (ylxS)

BS4050

-2.567

√

hypothetical protein (yybP)

BS2617

-2.559

√

hypothetical protein (yqaN)

BS0806

-2.555

√

acetoin dehydrogenase E1 component (TPP-dependent alpha subunit) (acoA)

BS0636

-2.553

√

GMP synthetase (guaA)

BS1871

-2.546

√

hypothetical protein (yoaS)

BS2152

-2.537

√

hypothetical protein (yolA)

BS1490

-2.535

√

cytochrome caa3 oxidase (subunit II) (ctaC)

BS3210

-2.518

transcriptional regulator (paiA)

BS0558

-2.516

√

hypothetical protein (ydgC)

BS1585

-2.516

similar to phosphoglycerate dehydrogenase (yloW)

BS3625

-2.513

transcriptional regulator (DeoR family) (glcR)

BS2512

-2.483

√

hypothetical protein (yqfN)

BS0164

-2.480

similar to transcriptional regulator (AraC/XylS family) (ybbB)

BS2895

-2.471

√

hypothetical protein (ytcF)

BS2632

-2.456

√

hypothetical protein (yrkS)

BS0804

-2.447

√

hypothetical protein (yfjM)

BS0632

-2.404

√

similar to cation efflux system membrane protein (yeaB)

BS2632

-2.392

√

hypotheitcal protein (yrkS)

BS2713

-2.387

√

similar to formate dehydrogenase (yrhG)

BS0561

-2.386

√

ATP-binding transport protein (expZ)

BS1373

-2.372

√

hypothetical protein (ykvJ)

BS0888

-2.371

√

hypothetical protein (ygaO)

BS2239

-2.369

ketopantoate hydroxymethyltransferase (panB)

BS2746

-2.356

√

hypothetical protein (yrvN)

BS3568

-2.347

UDP-glucose:polyglycerol phosphate glucosyltransferase (tagE)

BS4087

-2.342

√

similar to formate dehydrogenase (yyaE)

BS0745

-2.309

√

similar to quinone oxidoreductase (yfmJ)

BS3473

-2.308

√

similar to mutator MutT protein (yvcI)

BS2596

-2.308

√

similar to phage-related protein (yqbN)

BS1319

-2.296

√

cobalamin-independent methionine synthase (metC)

BS1472

-2.294

√

hypothetical protein (ylaA)

BS3265

-2.287

√

similar to ABC transporter (ATP-binding protein) (yurY)

BS3666

-2.250

required for formate dehydrogenase activity (narQ)

BS2225

-2.236

√

hypothetical protein (yppD)

BS3000

-2.232

√

hypothetical protein (ytfP)

BS1181

-2.230

√

hypothetical protein (yjcB)

BS1735

-2.220

hypothetical protein (ymzC)

BS0668

-2.210

√

hypothetical protein (yerL)

BS1065

-2.204

similar to DNA exonuclease (yirY)

BS3199

-2.201

√

hypothetical protein (yuiF)

BS1302

-2.182

hypothetical protein (ykgB)

BS2921

-2.181

hypothetical protein (ytoI)

BS3142

-2.171

√

similar to multidrug-efflux transporter (yuxJ)

BS2729

-2.163

√

similar to caffeoyl-CoA O-methyltransferase (yrrM)

BS0313

-2.157

hypothetical protein (ycgI)

BS3278

-2.157

similar to 3-hydroxyacyl-CoA dehydrogenase (yusL)

BS1386

-2.151

√

similar to heavy metal-transporting ATPase (ykvW)

BS0245

-2.139

√

similar to two-component sensor histidine kinase [YcbB] (ycbA)

BS3196

-2.124

√

hypothetical protein (yuiI)

BS0826

-2.115

√

similar to metabolite transport protein (yfiG)

BS1536

-2.115

√

similar to acetylornithine deacetylase (ylmB)

BS1142

-2.104

hypothetical protein (yjbA)

BS2376

-2.057

√

similar to pyrroline-5-carboxylate reductase (yqjO)

BS2920

-2.018

hypothetical protein (ytpI)

BS1878

-2.018

beta-lactamase precursor (penP)

First column is gene ID, second is Z score based on method in Materials and methods. Third column is checked if an attenuator has already been shown experimentally. Fourth column indicates if a promoter was predicted from NNPP program. Fifth column lists gene description. a. The NNPP program (cutoff = 0.8) was used for the upstream promoter prediction b GenBank annotation was used for gene definitions and gene names

In order to see if the observed patterns hold for the only other genome in which attenuation or antitermination is well studied and experimentally described, we also applied the same methodology to upstream regions of genes in the E. coli genome for which 16 operons have been described as being regulated by attenuation or antitermination. As can be seen in Figure 3, the known E. coli attenuation and antitermination terminator structures have similar properties as those of B. subtilis. 15 of the 16 known attenuators were maintained after filtering. The significance measure separates 14 of these E. coli terminators from random folds as seen in Figure 3. As in B. subtilis, using the (Z≤-2) line as a measure of significance, we are able to predict attenuation for 146 regions (Figure 3 and Table 2).

Figure 3

Stability and length distributions of stem-loop structures in upstream sequence segments in E. coli

Stability and length distributions of stem-loop structures in upstream sequence segments in E. coli. The red line shows the largest variance (see Materials and Methods) derived from stem-loop structures in shuffled sequences. Light blue lines give the significance measurements based on standard deviation. The definition for each point together with the orientation of neighboring genes are shown in upper right panel.

Table 2

Predicted attenuators in the genome of E. coli

Z-score

Known

Upstream promoter^a

Gene^b

b4119

-9.279

alpha-galactosidase (melA)

b2019

-7.560

√

ATP phosphoribosyltransferase (hisG)

b3828

-7.143

regulator for metE and metH (metR)

b3543

-7.009

dipeptide transport system permease protein 1 (dppB)

b4118

-6.911

regulator of melibiose operon (melR)

b2425

-6.168

√

thiosulfate binding protein (cysP)

b0560

-5.838

√

bacteriophage DNA packaging protein (nohB)

b4385

-5.755

hypothetical protein (yjjJ)

b3866

-5.703

hypothetical protein (yihI)

b0689

-5.697

putative pectinase (ybfP)

b1548

-5.638

√

homolog of Qin prophage packaging protein NU1 (nohA)

b1825

-5.573

√

hypothetical protein

b0611

-5.528

RNase I, cleaves phosphodiester bond between any two nucleotides (rna)

b2622

-5.470

prophage CP4-57 integrase (intA)

b3066

-5.002

DNA biosynthesis; DNA primase (dnaG)

b0654

-4.844

glutamate/aspartate transport system permease (gltJ)

b1978

-4.772

√

putative factor

b3767

-4.529

√

acetolactate synthase II, large subunit, cryptic, interrupted (ilvG_1)

b2119

-4.518

√

hypothetical protein (yehL)

b2792

-4.444

hypothetical protein

b0902

-4.443

√

pyruvate formate lyase activating enzyme 1 (pflA)

b3871

-4.352

√

putative GTP-binding factor (yihK)

b2937

-4.347

√

agmatinase (speB)

b0939

-4.300

putative chaperone (ycbR)

b1431

-4.280

√

hypothetical protein

b0890

-4.275

cell division protein (ftsK)

b2632

-4.177

√

putative GTP-binding protein (yfjP)

b0048

-4.168

√

dihydrofolate reductase type I (folA)

b0336

-4.133

cytosine permease/transport (codB)

b2517

-4.076

√

hypothetical protein (yfgB)

b3438

-4.018

regulator of gluconate (gnt) operon (gntR)

b2599

-3.945

√

chorismate mutase-P and prephenate dehydratase (pheA)

b3671

-3.923

√

acetolactate synthase I, valine-sensitive, large subunit (ilvB)

b2775

-3.907

√

putative transport protein (yqcE)

b2609

-3.891

√

30S ribosomal subunit protein S16 (rpsP)

b3181

-3.889

transcription elongation factor: cleaves 3' nucleotide of paused mRNA (greA)

b3983

-3.861

√

50S ribosomal subunit protein L11 (rplK)

b3722

-3.823

√

PTS beta-glucosides enzyme II, cryptic (bglF)

b0002

-3.788

√

aspartokinase I, homoserine dehydrogenase I (thrA)

b0196

-3.759

regulator in colanic acid synthesis; interacts with RcsB (rcsF)

b0007

-3.711

inner membrane transport protein (yaaJ)

b3513

-3.694

putative membrane protein (yhiU)

b3088

-3.669

√

putative transport protein (ygjT)

b4178

-3.630

√

hypothetical protein (yjeB)

b3752

-3.573

√

ribokinase (rbsK)

b2643

-3.572

hypothetical protein (yfjX)

b0074

-3.559

√

2-isopropylmalate synthase (leuA)

b2096

-3.530

√

tagatose-bisphosphate aldolase 1 (gatY)

b0170

-3.506

√

elongation factor EF-Ts (tsf)

b0610

-3.506

√

regulator of nucleoside diphosphate kinase (rnk)

b3813

-3.503

√

DNA-dependent ATPase I and helicase II (uvrD)

b1851

-3.446

6-phosphogluconate dehydratase (edd)

b2359

-3.395

√

hypothetical protein

b4245

-3.387

√

aspartate carbamoyltransferase, catalytic subunit (pyrB)

b4377

-3.374

√

hypothetical protein (yjjU)

b3298

-3.362

√

30S ribosomal subunit protein S13 (rpsM)

b0606

-3.358

alkyl hydroperoxide reductase, F52a subunit (ahpF)

b0018

-3.350

√

Gef protein interferes with membrane function when in excess (gef)

b2958

-3.329

hypothetical protein (yggN)

b3822

-3.303

ATP-dependent DNA helicase (recQ)

b0241

-3.292

outer membrane pore protein E (phoE)

b0680

-3.260

glutamine tRNA synthetase (glnS)

b0404

-3.233

putative glycoprotein (yajB)

b3337

-3.232

√

hypothetical protein (yheA)

b3190

-3.181

hypothetical protein (yrbA)

b0860

-3.147

arginine 3rd transport system periplasmic binding protein (artJ)

b1922

-3.115

√

flagellar biosynthesis; regulation of flagellar operons (fliA)

b2836

-3.103

2-acyl-glycerophospho-ethanolamine acyltransferase; acyl-acyl-carrier protein synthetase (aas)

b3915

-3.087

putative transport system permease protein (yiiP)

b3310

-3.083

√

50S ribosomal subunit protein L14 (rplN)

b0119

-3.018

hypothetical protein (yacL)

b3748

-3.005

D-ribose high-affinity transport system; membrane-associated protein (rbsD)

b2313

-2.990

√

membrane protein required for colicin V production (cvpA)

b1352

-2.967

√

hypothetical protein (ydaD)

b0441

-2.961

putative protease maturation protein (ybaU)

b3161

-2.947

tryptophan-specific transport protein (mtr)

b1614

-2.888

√

hypothetical protein (ydgA)

b3528

-2.862

√

uptake of C4-dicarboxylic acids (dctA)

b2514

-2.812

√

histidine tRNA synthetase (hisS)

b3390

-2.810

√

shikimate kinase I (aroK)

b1253

-2.808

hypothetical protein (yciA)

b2724

-2.808

√

probable small subunit of hydrogenase-3, iron-sulfur protein (part of formate hydrogenlyase (FHL) complex) (hycB)

b4242

-2.806

Mg2+ transport ATPase, P-type 1 (mgtA)

b0208

-2.804

putative transcriptional regulator LYSR-type (yafC)

b1479

-2.790

√

NAD-linked malate dehydrogenase (malic enzyme) (sfcA)

b1838

-2.749

√

protein phosphatase 1 modulates phosphoproteins, signals protein misfolding (pphA)

b0189

-2.741

√

hypothetical protein (yaeO)

b1264

-2.717

√

anthranilate synthase component I (trpE)

b4313

-2.706

√

recombinase involved in phase variation; regulator for fimA (fimE)

b3573

-2.691

hypothetical protein (yiaI)

b3642

-2.675

√

orotate phosphoribosyltransferase (pyrE)

b0034

-2.646

√

transcriptional regulator of cai operon (caiF)

b2176

-2.635

hypothetical protein (rtn)

b1769

-2.618

putative transport protein (ydjE)

b1561

-2.605

hypothetical protein (rem)

b1050

-2.605

hypothetical protein (yceK)

b2643

-2.604

hypothetical protein (yfjX)

b0248

-2.604

hypothetical protein (yafX)

b4278

-2.591

√

IS4 hypothetical protein (yi41)

b3560

-2.583

glycine tRNA synthetase, alpha subunit (glyQ)

b3723

-2.565

√

positive regulation of bgl operon (bglG)

b1689

-2.560

√

hypothetical protein

b3632

-2.528

√

lipopolysaccharide core biosynthesis (rfaQ)

b1404

-2.521

√

IS30 transposase (tra8_2)

b2014

-2.519

√

putative amino acid/amine transport protein (yeeF)

b2924

-2.487

putative transport protein (yggB)

b0070

-2.433

√

putative transport protein (yabM)

b4037

-2.409

√

periplasmic protein of mal regulon (malM)

b2359

-2.405

hypothetical protein

b3571

-2.393

√

alpha-amylase (malS)

b4082

-2.370

putative membrane protein (yjcR)

b0174

-2.360

√

hypothetical protein (yaeS)

b4392

-2.359

soluble lytic murein transglycosylase (slt)

b2938

-2.347

√

biosynthetic arginine decarboxylase (speA)

b2443

-2.331

√

hypothetical protein

b4090

-2.296

√

ribose 5-phosphate isomerase B (rpiB)

b2140

-2.289

√

putative regulator protein (yohI)

b1861

-2.274

√

Holliday junction helicase subunit B (ruvA)

b3196

-2.274

√

hypothetical protein (yrbG)

b3054

-2.268

hypothetical protein (ygiF)

b1761

-2.252

NADP-specific glutamate dehydrogenase (gdhA)

b0763

-2.248

√

molybdate-binding periplasmic protein; permease (modA)

b0909

-2.234

putative heat shock protein (ycaL)

b3954

-2.233

√

putative ARAC-type regulatory protein (yijO)

b3440

-2.232

√

putative regulator (yhhX)

b3074

-2.225

putative tRNA synthetase (ygjH)

b0605

-2.218

√

alkyl hydroperoxide reductase, C22 subunit; detoxification of hydroperoxides (ahpC)

b1053

-2.218

putative transport protein (yceE)

b1284

-2.210

√

putative DEOR-type transcriptional regulator

b0068

-2.206

thiamin-binding periplasmic protein (tbpA)

b0681

-2.204

hypothetical protein (ybfM)

b2150

-2.158

√

galactose-binding transport protein; receptor for galactose taxis (mglB)

b3349

-2.153

√

FKBP-type peptidyl-prolyl cis-trans isomerase (rotamase) (slyD)

b2298

-2.148

putative S-transferase (yfcC)

b0554

-2.133

√

hypothetical protein (ybcR)

b1778

-2.121

hypothetical protein (yeaA)

b1829

-2.092

√

heat shock protein, integral membrane protein (htpX)

b2685

-2.084

multidrug resistance secretion protein (emrA)

b2456

-2.069

detox protein (cchB)

b4284

-2.067

√

IS30 transposase (tra8_3)

b1002

-2.064

√

periplasmic glucose-1-phosphatase (agp)

b0854

-2.059

√

periplasmic putrescine-binding protein; permease protein (potF)

b1368

-2.051

putative alpha helix protein

b3088

-2.048

√

putative transport protein (ygjT)

b0347

-2.019

√

3-(3-hydroxyphenyl)propionate hydroxylase (mhpA)

b1663

-2.018

√

putative transport protein (ydhE)

First column is gene ID, second is Z score based on method in Materials and methods. Third column is checked if an attenuator has already been shown experimentally. Fourth column indicates if a promoter was predicted from NNPP program. Fifth column lists gene description. a The NNPP program (cutoff = 0.8) 26,27 was used for the upstream promoter prediction b GenBank annotation was used for gene definitions and gene names

Extension of analysis to 26 genomes

Analysis of B. subtilis and E. coli suggest that a broader survey of bacterial genomes might prove useful in both the prediction of attenuation and antitermination regulation in these genomes and the characterization of the evolution and distribution of these mechanisms of regulation. Twenty-four completed genomes were selected for this survey based on their broad distribution across the evolutionary spectrum (Table 3). The intergenic regions of each of these genomes were analyzed using the same methods and filters as with B. subtilis and E. coli and predicted attenuation and antitermination terminator folds similarly obtained.

Table 3

List of all 26 genomes surveyed in this study

Genome

No. of intergenic regions

No. of predicted attenuators

Archaea

Archaeoglobus fulgidus

1684

Methanococcus jannaschii

1515

Pyrococcus abyssi

1376

Eubacteria

Chlamydiales

Chlamydia pneumoniae

924

Cyanobacteria

Synechocystis sp.

2957

113

Gram-positive

Bacillus halodurans

3527

208

Bacillus subtilis

3650

203

Clostridium acetobutylicum

3386

275

Lactococcus lactis

1946

193

Listeria innocua

2565

154

Mycobacterium tuberculosis

3117

Mycoplasma genitalium

354

Staphylococcus aureus Mu50

2338

180

Streptococcus pneumoniae

1680

147

Proteobacteria (beta subdivision)

Neisseria meningitidis MC58

1868

118

Proteobacteria (gamma subdivision)

Buchnera sp.

550

Escherichia coli

3613

146

Haemophilus influenzae

1482

107

Pseudomonas aeruginosa

4756

Vibrio cholerae

2203

115

Xylella fastidiosa

2116

Proteobacteria (epsilon subdivision)

Campylobacter jejuni

1028

Helicobacter pylori J99

1150

Spirochaetales

Borrelia burgdorferi

638

Thermas/Deinococcus

Deinococcus radiodurans

2055

Thermotogales

Thermotoga maritima

1173

As shown in Table 3, there is a wide distribution of the number of putative attenuation and antitermination regulatory sites in the surveyed genomes. These range from 5 in Mycobacterium tuberculosis to 275 in Clostridium acetobutylicum (Table 3). Earlier attempts to predict standard transcription termination sites at the end of transcription units give similar results. Interestingly, the results for standard transcription terminators correlate with ours. As was found in Ermolaeva et. al [13] with standard terminators at the end of transcription units (this paper studied terminators at end of ORFs and did not target upstream regions, thus filtering out possible attenuators), some of the highest number of occurrences of attenuation and antitermination sites in our survey are similarly found in the genomes of E. coli, H. influenze, D. radiodurans and B. subtilis and the lowest number of occurrences in such genomes as H. pylori, and M. tuberculosis (genomes reported in their survey).

At first glance, this would seem to suggest that many genomes do not use the same mechanisms of termination for the standard transcription termination and do not use attenuation or antitermination in regulation. This is likely the case in some genomes. Yet, if the number of upstream intergenic regions is plotted against the number of predicted sites, a strong positive correlation is shown (Figure 4). The smaller the number of genes and intergenic regions a genome has, the lower the occurrence of predicted terminators (both standard transcription terminators and attenuation/antitermination regulatory terminators). This indicates that the low numbers of both standard termination and regulatory termination in many genomes is due to a much reduced genome size and the reduction of the number of regulatory operons, and not necessarily to the reliance on different mechanisms of termination and regulation.

Figure 4

Graph of the number of intergenic regions vs. the number of putative attenuation and antitermination sites in all 26 genomes surveyed

Graph of the number of intergenic regions vs. the number of putative attenuation and antitermination sites in all 26 genomes surveyed. Several genomes with known attenuation or antitermination are labeled for comparison as is M. tuberculosis and the Archaea. The dashed line is a exponential trendline.

There is a clear outlier with a much lower than expected number of putative terminators seen in Figure 4, Mycobacterium tuberculosis. This genome has a much lower occurrence of putative attenuation and antitermination sites than would be suggested by its size and the number of intergenic regions. A recent paper by Unniraman et al. [18] concludes that M. tuberculosis uses a different mechanism of termination that utilizes terminator structures without the poly-U tail necessary in other genomes. Thus the reduced number of poly-U containing terminator structures in relation to the number of intergenic regions can be explained by M. tuberculosis' reliance on a different mechanism of termination. This does not necessarily prove there is no attenuation or antitermination type regulation in M. tuberculosis. However, it does indicate that either the loss of the standard mechanism of termination in this genome has reduced if not eliminated attenuation or antitermination in M. tuberculosis or alternatively, an attenuation-like mechanism could exist in this genome that utilizes the M. tuberculosis non-standard terminator.

All other of the 25 genomes surveyed have putative attenuation or antitermination regulation sites. Even the lowest number of predicted attenuation or antitermination sites found in M. genitalium are a significant proportion of possible regulatory intergenic regions, the low number is easily accounted for by this genome's relatively small size and few intergenic regions and transcriptional units. These results suggest that attenuation and antitermination regulation is a possibly ubiquitous mechanism of regulation in prokaryotes with few exceptions.

Genome Size and Attenuation

If the GC content of a genome is compared with the number of predicted attenuators based on randomly shuffled sequence, GC content does somewhat correlate with the number of predicted attenuators, which would be expected since a poly-U run is required in the filters. In Figure 5a, folds from randomly shuffled intergenic sequences of our 26 genomes were plotted by the number of filtered folds per intergenic region in relation to number of intergenic regions. If the number of filtered folds was completely random, there should be a relatively constant number of sites per region in relation to the number of regions. As seen in figure 5a, this is not completely the case. The number of filtered folds per region obtained from randomly shuffled sequences is dependent on the GC content of the genome. Low-GC content genomes have a slightly higher per region number of folds than do genomes of around 50% GC content and high-GC content genomes have much lower number than both. This is expected from random sequences filtered for stem-loop structures containing poly-U runs.

Figure 5

Genome Size and Regulation

Genome Size and Regulation. (a) Intergenic sequences of 26 genomes were randomly shuffled, folded and filtered using reported method to obtain putative 'attenuators'. The number of these shuffled and filtered folds per intergenic region were plotted for each genome against the number of intergenic regions. The correlation, if random, should remain constant and independent of genome size. Blue spheres represent proteobacteria and Bacillis species in our survey, beige are archaeabacteria and green the rest. Spheres are in size in proportion to the genome's GC content and GC content is labeled within each sphere. The number of random folds per intergenic region is a function of GC content as would be expected from filtering for folds with poly-U runs. Genomes with known attenuation or antitermination are labeled as is the genome known not to use attenuators with poly-U runs in termination. (b) Intergenic sequences of 22 genomes were folded and filtered for possible attenuators and indication of attenuation or antitermination regulation. The number of these predicted attenuators per intergenic region is compared to the number of intergenic regions in the genome. In contrast to folds of randomly shuffled sequences, the strongest determinate for the frequency of attenuation is genome size (number of intergenic regions and genome size are strongly correlated). Colors and labeling are the same as in 5a.

Even when taking into account the GC content of M. tuberculosis, it has a reduced number of predicted attenuators in relation to the other high-GC genomes (Figure 5b). In fact, Figure 5b (predicted attenuators of actual intergenic sequences) shows that the strongest determinate of the number of predicted attenuators per intergenic region is not GC content but rather genome size (more specifically the number of intergenic regions). In general, not only do larger genomes have a greater absolute number of predicted attenuators, but have a greater occurrence of predicted attenuators per region. If GC content is equal in two genomes, the larger genome is more likely to have a higher number of predicted attenuators per intergenic region. Previous reports have suggested similar phenomena in regulatory proteins, large genomes appear to have a larger proportion of their total number of genes that code for proteins which contain regulatory motifs [19]. Interestingly, discounting the archaebacteria and high GC content genomes, a genome of about 1500 intergenic regions appears to be the threshold at where the frequency of regulatory attenuators increases in a genome.

Distribution and Conservation of Attenuators in Gram positive Bacteria

Seven genomes of gram-positive bacterias (B. subtilis, B. halodurans, L. innocua, S. aureus, C. acetobutylicum, L. lactis, and S. pneumoniae) were analyzed to see whether the attenuation terminators are conserved in front of the orthologs. The number of predicted attenuation terminators for the genes known to be regulated in B. subtilis and their orthologs in the other six genomes are listed in Table 4. The genomes are sorted by phylogenetic distance from B. subtilis calculated by amino acid sequences of the shared orthologs among these genomes. The closest one to the B. subtilis is B. halodurans and the averaged number of amino acid substitutions per site is 0.238, and the most distant one is S. pneumoniae and the averaged number of amino acid substitutions per site is 0.422. For the 42 genes listed in Table 4, the numbers of orthologs that are found in the other genomes vary little from genome to genome: The highest and the lowest numbers of orthologs are 31 in L. lactis and 26 in S. aureus and C. acetobutylicum, respectively. This is mainly because these 42 genes carry some basic functions such as aminoacyl-tRNA synthesis. On the other hand, the numbers of predicted attenuation termination structures vary significantly: In B. halodurans, 22 orthologous genes have predicted attenuation termination structures, while only 4 orthologous genes have the predicted structures in S. pneumoniae. This indicates that the absence or presence of regulation by attenuation is much more weakly conserved than the gene or operons presence.

Table 4

List of known attenuators in B. subtilis compared with predictions in six other genomes of gram-positive bacteria

B. subtilis

B. halodurans

L. innocua

S. aureus

C. acetobutylicum

L. lactis

S. pneumoniae

Z-score

ID^a

No. of predictions^b

ID^a

No. of predictions^b

ID^a

No. of predictions^b

ID^a

No. of predictions^b

ID^a

No. of predictions^b

ID^a

No. of predictions^b

Genec

BS0929

-8.895

BH1095

lin1331

SAV1302

L0013

SP2185

glycerol-3-phosphate dehydrogenase

(glpD)

BS2825

-8.473

BH3061

lin2091

SAV2054

CAC3169

L0078

SP0445

acetolactate synthase (large subunit)

(ilvB)

BS3920

-6.468

BH0296

lin0026

CAC1407

L90678

SP0577

PTS beta-glucoside-specific enzyme

II ABC component (bglP)

BS2733

-6.238

BH1267

lin1539

SAV1618

CAC1678

L0343

SP1383

alanyl-tRNA synthetase (alaS)

BS0013

-6.126

BH0024

lin2890

SAV0009

CAC0021

L150515

SP0411

seryl-tRNA synthetase (serS)

BS2749

-5.839

BH1251

lin1555

SAV1631

CAC2740

L0342

SP2121

histidyl-tRNA synthetase (hisS)

BS2520

-5.815

BH1370

lin1496

L101560

SP1475

glycyl-tRNA synthetase (alpha

subunit) (glyQ)

BS3396

-5.767

L18622

amino acid permease (yvbW)

BS0215

-5.526

SAV0337

L148346

glycerol-3-phosphate permease

(glpT)

BS1544

-5.239

BH2545

lin2127

SAV1193

CAC3038

L0350

SP1659

isoleucyl-tRNA synthetase (ileS)

BS3798

-5.164

BH1856

CAC0423

PTS sucrose-specific enzyme II BC

component (sacP)

BS1319

-5.124

BH0438

lin1789

SAV0356

L0100

SP0585

cobalamin-independent methionine

synthase (metC)

BS3750

-4.958

CAC2362

threonyl-tRNA synthetase (thrZ)

BS3440

-4.890

CAC1772

levansucrase (sacB)

BS2204

-4.819

BH1514

lin1998

SAV0388

CAC0873

L159396

SP1847

xanthine phosphoribosyltransferase

(xpt)

BS2139

-4.756

N-acetylmuramoyl-L-alanine amidase

(yomC)

BS2803

-4.750

BH3038

lin1587

SAV1663

CAC2399

L0351

SP0568

valyl-tRNA synthetase (valS)

BS0093

-4.747

BH0110

lin0270

SAV0529

CAC0687

L0087

SP0589

serine acetyltransferase (cysE)

BS1357

-4.680

hypothetical protein (ykrT)

BS3839

-4.472

BH3228

CAC0780

tyrosyl-tRNA synthetase (tyrZ)

BS2961

-4.430

lin1639

SAV1729

CAC0637

L0359

SP2100

tyrosyl-tRNA synthetase (tyrS)

BS1143

-4.382

BH2870

lin2301

SAV0996

CAC0626

L0358

SP2229

tryptophanyl-tRNA synthetase (trpS)

BS0254

-4.309

hypothetical protein (yczA)

BS0643

-4.279

BH0623

lin1887

SAV1064

CAC1390

L152487

SP0053

phosphoribosylaminoimidazole

carboxylase I (purE)

BS3026

-4.270

BH3281

lin1769

SAV1760

CAC0646

L0352

SP0254

leucyl-tRNA synthetase (leuS)

BS2889

-4.249

BH3141

lin1594

SAV1683

L0357

SP1631

threonyl-tRNA synthetase (thrS)

BS1188

-4.176

BH1627

lin1788

SAV0359

L0102

SP1525

cystathionine gamma-synthase (yjcI)

BS1313

-4.086

BH1505

L0117

SP0931

gamma-glutamyl kinase (proB)

BS3269

-3.989

BH3481

lin2514

SAV0837

CAC0984

L121289

SP0151

ABC transporter (ATP-binding

protein) (yusC)

BS3900

-3.884

BH3232

CAC2807

endo-beta-1,3-1,4 glucanase (bglS)

BS0038

-3.883

BH0053

lin0216

SAV0490

CAC2991

L0353

SP0788

methionyl-tRNA synthetase (metS)

BS2858

-3.784

BH3111

lin1184

SAV1138

CAC2357

L0354

SP0579

phenylalanyl-tRNA synthethase

(alpha subunit) (pheS)

BS0927

-3.776

BH1092

lin1574

SAV1300

CAC1319

L0015

SP2184

glycerol uptake facilitator (glpF)

BS1548

-3.473

BH2541

lin1954

SAV1198

CAC2113

L0227

SP1278

uracil phosphoribosyltransferase

(pyrR)

BS3888

-3.412

lin0838

L124252

hypothetical protein (yxjH)

BS3889

-3.412

hypothetical protein (yxjG)

BS1549

-3.265

BH2540

lin1953

SAV1199

CAC2112

L46118

SP1286

uracil permease (pyrP)

BS1550

-3.120

BH2539

lin1952

SAV1200

CAC2654

L45002

SP1277

aspartate carbamoyltransferase

(pyrB)

BS1166

-3.059

BH2679

lin0340

SAV2094

L0228

SP0722

transcriptional regulator (tenA)

BS1360

-2.966

ribulose-bisphosphate carboxylase

(ykrW)

BS1855

-2.649

phosphoglycerate dehydrogenase

(yoaD)

BS2376

-2.057

BH1503

lin0414

SAV1503

CAC3252

L135991

SP0933

pyrroline-5-carboxylate reductase

(yqjO)

a "-" signifies that the absence of an ortholog. b "-" signifies that no prediction is made because of absence of ortholog c Gen back annotation of B. subtilis was used for gene definitions and gene names

The same trend holds true for the predicted attenuation termination structures other than known ones (Table 5). There are 105 orthologous gene groups that have at least one other genome containing a predicted attenuator structure upstream an orthologous gene. Restricting to the orthologs that have predicted attenuators in B. subtilis (35 groups), the highest and the lowest numbers of shared orthologs of genes known to be regulated by attenuation or antitermination in B. subtilis are 28 (L. innocua) and 18 (S. pneumoniae), respectively. The numbers of predicted attenuation termination structures, however, vary more. While there are 13 genes with predicted structures in B. halodurans, which is the closest species to B. subtilis among the six gram-positive bacterias, only 2 genes have predicted structures in S. pneumoniae.

Table 5

List of all orthologous genes in the six gram-positive bacteria genomes in which two or more genomes share predicted attenuators

B. subtilis

B. halodurans

L. innocua

S. aureus

C. acetobutylicum

L. lactis

S. pneumoniae

ID^a

Z-score^b

ID^a

No. of predictions^c

ID^a

No. of predictions^c

ID^a

No. of predictions^c

ID^a

No. of predictions^c

ID^a

No. of predictions^c

ID^a

No. of predictions^c

Gene^d

BS1310

-6.836

BH0686

lin0846

chaperonin (ykkC)

BS3710

-5.196

BH3792

lin2704

SAV2127

CAC2892

L88252

SP0494

CTP synthetase (ctrA)

BS2484

-5.148

BH1417

lin1373

SAV1550

CAC1090

L172782

SP2095

5-formyltetrahydrofolate cyclo-ligase

(yqgN)

BS0183

-4.938

SAV0452

NADH dehydrogenase (subunit 5)

(ndhF)

BS3877

-4.696

lin1219

SAV0646

hypothetical protein (yxkD)

BS0535

-4.505

BH2084

antibiotic resistance protein (ydfB)

BS2452

-4.143

BH2816

lin1385

SAV1537

aminomethyltransferase (yqhI)

BS0143

-3.709

BH0162

lin2755

SAV2224

CAC3104

L0136

SP0236

RNA polymerase (alpha subunit)

(rpoA)

BS0104

-3.612

BH0121

lin0282

SAV0539

CAC3146

L0407

SP1355

ribosomal protein L10 (rplJ)

BS0002

-3.480

BH0002

lin0002

SAV0002

CAC0002

L0275

SP0002

DNA polymerase III (beta subunit)

(dnaN)

BS2881

-3.464

BH3140

lin1897

SAV1680

CAC2361

L95240

SP0959

initiation factor IF-3 (infC)

BS3093

-3.325

BH0830

lin1468

CAC2928

L116212

hypothetical protein (yuaJ)

BS2841

-3.262

BH3096

lin1198

SAV1393

aspartokinase II (lysC)

BS2903

-3.222

BH3153

lin1600

SAV1690

CAC1098

L0270

SP0032

DNA polymerase I (polA)

BS1920

-3.201

lin2900

SAV0721

CAC2687

L0268

ATP-dependent DNA helicase (yocI)

BS3731

-3.192

CAC2795

L19128

hypothetical protein (ywiA)

BS2278

-3.156

BH1643

hypothetical protein (yphE)

BS0878

-3.120

BH1933

CAC3014

thiamin biosynthesis protein (thiA)

BS2539

-3.075

BH1348

lin1509

SAV1579

CAC1283

L0272

SP0519

heat-shock protein (dnaJ)

BS1659

-2.908

BH2417

lin1358

SAV1265

CAC1798

L173151

SP0552

hypothetical protein (ylxS)

BS0115

-2.894

BH0133

lin2782

SAV2251

CAC3134

L0387

SP0208

ribosomal protein S10 (rpsJ)

BS0637

-2.878

BH0608

lin0582

SAV2253

CAC2772

hypothetical protein (yebB)

BS2627

-2.867

lin0073

SAV1098

transcriptional regulator (yqaE)

BS2986

-2.643

BH3263

lin1657

SAV1749

L155396

SP0549

hypothetical protein (ytmP)

BS1659

-2.573

BH2417

lin1358

SAV1265

CAC1798

L173151

SP0552

hypothetical protein (ylxS)

BS0636

-2.553

BH0607

lin1081

SAV0391

CAC2700

L115968

SP1445

GMP synthetase (guaA)

BS0558

-2.516

lin1827

L53789

hypothetical protein (ydgC)

BS2512

-2.483

BH1379

lin1490

SAV1560

CAC1302

L84257

SP1610

hypothetical protein (yqfN)

BS0561

-2.386

CAC3007

ATP-binding transport protein (expZ)

BS3473

-2.308

BH3570

lin1215

SAV0467

CAC0084

L0288

SP0740

mutator protein (yvcI)

BS1319

-2.296

BH0438

lin1789

SAV0356

L0100

SP0585

cobalamin-independent methionine

synthase (metC)

BS3265

-2.287

BH3471

lin2510

SAV0842

CAC3288

L34806

SP0867

ABC transporter (ATP-binding

protein) (yurY)

BS0668

-2.210

BH0665

lin1868

SAV1901

CAC2671

L0475

SP0438

glutamyl-tRNA(Gln)

amidotransferase (subunit C) (yerL)

BS2729

-2.163

BH1272

lin1533

SAV1614

CAC1686

L165684

SP0980

O-methyltransferase (yrrM)

BS1386

-2.151

BH0744

lin0644

L63697

heavy metal-transporting ATPase

(ykvW)

BS0019

BH0034

lin2852

SAV0478

CAC0125

L0279

SP0865

DNA polymerase III (gamma and tau

subunits) (dnaX)

BS0106

BH0124

lin0284

SAV0541

CAC0877

L65498

SP0841

hypothetical protein (ybxB)

BS0107

BH0126

lin0285

SAV0542

CAC3143

L0137

SP1961

DNA-directed RNA polymerase (beta

subunit) (rpoB)

BS0112

BH0131

lin2803

SAV0547

CAC3138

L0368

SP0273

elongation factor G (fus)

BS0462

BH0518

lin0884

SAV2071

CAC0489

L61355

SP1699

acyl carrier protein synthase (ydcB)

BS0516

BH0725

SAV2534

CAC0875

L124727

SP1447

hypothetical protein (ydeD)

BS0663

BH0649

lin1870

SAV1904

CAC2673

L0304

SP1117

DNA ligase (yerG)

BS0692

lin0655

SAV1419

CAC2751

L36177

SP1464

hypothetical protein (yesJ)

BS0824

BH3305

lin0649

SAV2522

SP0073

hypothetical protein (yfiE)

BS0892

BH1023

SAV1855

CAC0700

L161988

SP0486

rRNA methylase (cspR)

BS0960

BH2987

SAV1783

CAC1586

SP1295

hypothetical protein (yhdV)

BS0988

BH0202

lin1781

SAV1045

CAC2712

L0171

SP0415

similar to 3-hydroxybutyryl-CoA

dehydratase (yhaR)

BS1062

lin2369

SAV0966

CAC2263

L0252

SP1151

ATP-dependent deoxyribonuclease

(subunit B) (addB)

BS1139

BH3636

lin0182

SAV0991

CAC3179

L88446

oligopeptide ABC transporter

(oligopeptide-binding protein) (appA)

BS1269

BH0012

lin2568

SAV1955

CAC1883

L60836

prophage (xkd0)

BS1345

BH2553

lin0990

SAV1022

CAC1415

similar to toxic anion resistance

protein (ykoY)

BS1390

BH0844

SAV0189

CAC0570

SP1684

PTS glucose-specific enzyme II ABC

component (ptsG)

BS1478

BH2632

lin1055

SAV1109

CAC1684

L0370

SP0681

similar to GTP-binding elongation

factor (ylaG)

BS1512

BH2579

lin2152

SAV2443

CAC2937

L157055

similar to ketopantoate reductase

(ylbQ)

BS1514

BH2576

lin2148

SAV1178

CAC2133

hypothetical protein (yllB)

BS1553

BH2536

lin1949

SAV1203

CAC2644

L198033

SP1275

carbamoyl-phosphate synthase

(catalytic subunit) (pyrAB)

BS1566

BH2515

lin0832

CAC2137

L2866

SP1551

cation-transporting ATPase (yloB)

BS1570

BH2510

lin1939

SAV1211

CAC1720

L166912

SP1231

pantothenate metabolism flavoprotein

(yloI)

BS1587

BH2495

lin1925

SAV1227

CAC1736

L0262

SP1697

ATP-dependent DNA helicase recG

(ylpB)

BS1614

BH1529

lin1316

SAV1252

CAC2066

L34517

SP0890

integrase/recombinase (codV)

BS1650

BH2426

lin1766

SAV1257

CAC1788

L0376

SP2214

elongation factor Ts (tsf)

BS1653

BH2423

lin1352

SAV1260

CAC1791

L183602

SP0261

undecaprenyl diphosphate synthetase

(yluA)

BS1668

BH2408

lin1367

SAV1273

CAC1807

L0392

SP1626

ribosomal protein S15 (rpsO)

BS1669

BH2407

lin1368

SAV1274

CAC1808

L0325

SP0588

polyribonucleotide phosphorylase

(pnpA)

BS1677

BH1742

lin1474

SAV1395

CAC2378

L0093

SP1014

dihydrodipicolinate synthase (dapA)

BS2023

BH3508

CAC1501

SP1336

DNA-methyltransferase (Bsu) (mtbP)

BS2185

BH3062

lin2090

SAV2053

CAC3170

L0077

SP2126

dihydroxy-acid dehydratase (ilvD)

BS2258

BH1665

lin2039

SAV0724

CAC3031

L0065

histidinol-phosphate aminotransferase

(hisC)

BS2264

BH1659

lin1674

SAV1367

CAC3163

L0054

SP1817

anthranilate synthase (trpE)

BS2301

lin2059

SAV1485

CAC2841

L106755

SP0488

hypothetical protein (ypaA)

BS2324

BH1554

SAV1771

CAC0590

L0163

SP0178

riboflavin-specific deaminase (ribG)

BS2334

BH1544

lin2066

SAV1400

CAC0608

L0121

SP1978

diaminopimelate decarboxylase (lysA)

BS2383

BH1472

lin2082

SAV1895

CAC0285

L0305

SP0458

DNA-damage inducible protein

(yqjH)

BS2661

SAV1407

CAC1610

SP0626

branched-chain amino acid

transporter (brnQ)

BS2741

BH1262

lin1545

SAV1620

CAC1067

hypothetical protein (yrrB)

BS2747

BH1255

SAV1628

CAC0908

SP0695

hypothetical protein (yrvM)

BS2932

BH0170

lin2443

SAV2414

CAC3325

L162009

SP0148

similar to amino acid ABC

transporter (binding protein) (ytmJ)

BS2942

BH3193

lin1617

SAV1712

L74738

SP2045

hypothetical protein (ytxK)

BS3015

BH0783

SAV2427

CAC1361

dethiobiotin synthetase (bioD)

BS3049

BH3300

lin1773

SAV1790

CAC2856

L153408

SP0762

S-adenosylmethionine synthetase

(metK)

BS3345

BH0557

lin1967

SAV2557

CAC3655

L45966

SP0729

heavy metal-transporting ATPase

(yvgX)

BS3388

BH3559

lin2552

SAV0773

CAC0710

L0010

SP0499

3-phosphoglycerate kinase (pgk)

BS3496

BH0421

lin0955

SAV0701

CAC0188

L173068

SP2056

N-acetylglucosamine-6-phosphate

deacetylase (nagA)

BS3705

BH3784

lin2697

SAV2124

CAC3539

L113067

SP1081

UDP-N-acetylglucosamine 1-

carboxyvinyltransferase (murZ)

BS3728

BH0834

lin2706

SAV0607

CAC1041

L0344

SP2078

arginyl-tRNA synthetase (argS)

BS3729

BH3809

lin2707

SAV0606

CAC2894

hypothetical protein (ywiB)

BS3875

BH1141

lin2875

CAC3236

L26721

SP0593

hypothetical protein (yxkF)

BS3901

BH0297

lin2530

SAV1357

CAC0422

L0154

SP0576

transcriptional antiterminator (BglG

family) (licT)

BS3916

lin0454

CAC1057

cell wall-associated protein precursor

(wapA)

BS3918

BH3184

lin1615

SAV1710

L84477

SP1996

hypothetical protein (yxiE)

BS3919

lin0344

CAC0743

SP0578

beta-glucosidase (bglH)

BS4099

BH4065

lin2987

SAV2713

CAC3738

L131443

SP2042

ribonuclease P (protein component)

(rnpA)

BH0440

SAV0142

L97415

phosphonate ABC transporter ATP-

binding protein

BH0503

CAC2487

L3279

SP0204

hypothetical protein

BH0595

lin0026

CAC1407

L90678

PTS beta-glucoside-specific enzyme

II ABC component

BH2025

lin2903

ABC transporter (ATP-binding

protein)

BH2200

L0358

SP2229

tryptophanyl-tRNA synthetase

BH2603

lin0604

CAC2783

L75975

O-acetylhomoserine sulfhydrylase

CAC1072

L180742

hypothetical protein

L49741

SP1647

endopeptidase