Comparative genomic analysis of two-component regulatory proteins inPseudomonas syringae

  • José L Lavín1,

    Affiliated with

    • Kristoffer Kiil2,

      Affiliated with

      • Ohiana Resano1,

        Affiliated with

        • David W Ussery2 and

          Affiliated with

          • José A Oguiza1Email author

            Affiliated with

            BMC Genomics20078:397

            DOI: 10.1186/1471-2164-8-397

            Received: 27 April 2007

            Accepted: 31 October 2007

            Published: 31 October 2007

            Abstract

            Background

            Pseudomonas syringaeis a widespread bacterial plant pathogen, and strains ofP. syringaemay be assigned to different pathovars based on host specifiCity among different plant species. The genomes ofP. syringaepv.syringae(Psy) B728a, pv.tomato(Pto) DC3000 and pv.phaseolicola(Pph) 1448A have been recently sequenced providing a major resource for comparative genomic analysis. A mechanism commonly found in bacteria for signal transduction is the two-component system (TCS), which typically consists of a sensor histidine kinase (HK) and a response regulator (RR).P. syringaerequires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions.

            Results

            Based on the genomic data, pattern searches with Hidden Markov Model (HMM) profiles have been used to identify putative HKs and RRs. The genomes ofPsyB728a,PtoDC3000 andPph1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed important differences in TCS proteins among the threeP. syringaepathovars.

            Conclusion

            In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes ofP. syringae. We have identified differences in TCS proteins among the threeP. syringaepathovars that may contribute to their diverse host ranges and association with plant hosts. The identification and analysis of the repertoire of TCS proteins in the genomes ofP. syringaepathovars constitute a basis for future functional genomic studies of the signal transduction pathways in this important bacterial phytopathogen.

            Background

            Bacterial signal transduction pathways sense the cellular external environment and regulate cellular functions in response to environmental signals. A mechanism commonly found in bacteria for signal transduction is the two-component system (TCS). Bacterial TCSs are common components of complex regulatory networks and cascades, often associated with global regulation as well as with regulation of virulence. TCS genes are typically located within the same operon encoding two signalling proteins: a transmembrane sensor histidine kinase (HK) and a cytoplasmic response regulator (RR), which may sometimes be carried by a single polypeptide to form the hybrid HKs [1]. The mechanism of signal transduction by TCS proteins is based on phosphotransfer reactions between histidine (H) and aspartate (D) residues in highly conserved signalling domains of the HKs and their cognate RRs. TCS proteins have a modular organization, which may give rise to highly complex structures, but the core structures and activities are maintained [2]. HKs are typically organized as homodimers with two functionally and structurally distinct domains: a highly variable N-terminal extracytoplasmic sensory domain, and a more conserved C-terminal cytoplasmic transmitter domain, also known as the dimerization/phosphoacceptor domain [2,3]. The sensor domain varies in length and amino acid sequence from one HK to another, conferring specifiCity for different environmental stimuli. In most HKs, the transmitter domain shows high sequence conservation, especially within a set of six recognizable motifs or boxes designated H, N, F, G1, G2, and G3. In particular, the H box contains an invariant H residue that is autophosphorylated in an ATP-dependent manner [4]. In contrast, CheA-like HKs that function in chemotaxis lack the sensor domain and differ from other HKs in their domain constitution and organization, where the H box of the transmitter domain resides at the N-terminal end of the protein [58]. LytS-like HKs also differ significantly in their domain architecture from other HKs [911]. RRs generally contain at least two functional domains: a conserved N-terminal receiver domain (REC domain) that is phosphorylated by the HK at a strictly conserved D residue, and one or more variable C-terminal output domains [12]. Modulation of the phosphorylated state of the RR controls either expression of the target genes or cellular behaviour. The principal type of bacterial RRs are transcription factors that regulate gene-expression with DNA-binding helix-turn-helix (HTH) output domains [1,3,12,13]. Hybrid HKs contain both a HK transmitter domain and a REC domain in a single large polypeptide, and are characterized by multi-step phosphotransfer reactions [1,7,14].

            The availability of complete genome sequences for a continually growing number of bacteria has allowed the definitive assessment that TCS proteins are present in almost all bacterial species [1,8,12]. Genomic analyses demonstrate the enormous impact of TCSs on environmental adaptation of bacteria, and reveal a wide variation of HK and RR numbers between different bacterial species [7,8,12,1520].

            The bacterial plant pathogenPseudomonas syringaecauses disease on a variety of plant species, and strains ofP. syringaehave been classified into different pathovars depending on their host range among different plant species [21]. Infection of host plants byP. syringaeinvolves growth on leaf surfaces as an epiphyte, that enters plant leaves through stomata, multiplies to large populations in the apoplast and produces disease symptoms [21,22].P. syringaeinjects effector proteins into the cytoplasm of plant cells by means of the Hrp type III secretion system [21]. Genome comparisons indicate thatP. syringaeis significantly different from otherPseudomonasspecies [23,24], suggesting that in the adaptation to the phytopathogenic lifestyle its genome must have undergone fundamental changes without a reduction in size. The complete genomic sequences of three economically important pathovars of this plant pathogenic bacteria have been determined: P. syringaepv.tomato(Pto) DC3000, pv.syringae(Psy) B728a and pv.phaseolicola(Pph) 1448A [2527]. In these genomes, over 10 to 12 % of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth, plant colonization and pathogenesis [2527]. Genome analyses of theseP. syringaepathovars revealed fewer extracytoplasmic function (ECF) sigma factors (10 ECF sigma factors) than in relatedPseudomonaswith different lifestyles [24]. Recently, analysis of thePtoDC3000 genome sequence allowed the identification of 69 HKs [28,29] and 71 RRs, 21 of which were hybrid HKs [12]. In a different study not including CheA-like HKs, 64 HKs were identified inPtoDC3000, 20 of which were hybrid HKs [30]. Hence,P. syringaerequires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions. The availability of complete genomic sequences of three differentP. syringaepathovars makes it possible to conduct this comparative genomic study to identify and analyse the TCS proteins ofP. syringae.

            Results and Discussion

            Distribution of TCS proteins inP. syringae

            The putative HKs and RRs inPsyB728a,PtoDC3000 andPph1448A were identified by searching the complete genome sequences for proteins containing HK and RR domains using Pfam HMM profiles. Four CheA-like HKs in eachP. syringaegenome were identified in BLASTP searches using as template the CheA HK ofE. coli[31] (Table 1). In addition, BLASTP searches of the HKs and RRs found in eachP. syringaepathovar against the genomes of the other two pathovars allowed the identification of additional HKs and RRs. The genomes ofP. syringaepathovars were found to contain large numbers of genes encoding TCS proteins: 68 HKs and 93 RRs inPsyB728a, 69 HKs and 95 RRs inPtoDC3000, and 70 HKs and 92 RRs inPph1448A (Table 1; see Additional File1and2). The number of genes encoding hybrid HKs (REC-HKs) was 20 inPsyB728a, 22 inPtoDC3000 and 24 inPph1448A (Tables 1and 4). The HMM search method used in this work retrieved hybrid HKs as well as RRs (Table 1). No TCS proteins were identified on any of the plasmids ofPtoDC3000 andPph1448A. In recent studies, similar numbers of TCS proteins forPtoDC3000 have been reported: 69 HKs [28,29] and 71 RRs, 21 of which were hybrid HKs [12]; or 64 HKs in a study not including CheA-like HKs, 20 of which where hybrid HKs [30]. Although the number of ECF sigma factors in all threeP. syringaegenomes (10 ECF sigma factors) is only about half that found in otherPseudomonasspecies [24,32], the number of TCS proteins is close to that found in otherPseudomonasgenomes [33].
            Table 1

            Distribution of HKs and RRs found in the genomes ofP. syringaepv.syringaeB728a, pv.tomatoDC3000 and pv.phaseolicola1448A.

            HK type/RR type

            P. syringaepv.syringaeB728a

            P. syringaepv. tomato DC3000

            P. syringaepv.phaseolicola1448A

            Histidine kinases

               

            Type IA

            22

            20

            21

            Type IB

            13

            15

            15

            Type IC

            20

            21

            22

            Type III

            2

            2

            2

            CheA-like

            4

            4

            4

            GAF-HK

            6

            6

            5

            LytS-like

            1

            1

            1

            Total HKs

            68

            69

            70

            Response regulators

               

            Stand-alone REC

            12

            13

            10

            OmpR-like

            22

            20

            19

            NarL-like

            9

            12

            10

            NtrC-like

            11

            11

            11

            LytR-like

            2

            2

            2

            PrrA-like

            1

            1

            1

            PleD-like

            5

            4

            5

            RsbU-like

            2

            2

            2

            CheB-like

            3

            3

            3

            CheC-like

            1

            1

            1

            CheW-like

            2

            2

            2

            VieA-like

            1

            1

            1

            VieB-like

            1

            1

            1

            AmiR-like

            1

            --

            --

            REC-HK (hybrid HK)

            20

            22

            24

            Total RRs

            93

            95

            92

            Table 4

            Hybrid HK genes in the genomes ofP. syringaepv.syringaeB728a, pv.tomatoDC3000 and pv.phaseolicola1448A.

            P. syringaepv.syringaeB728a

            P. syringaepv. tomato DC3000

            P. syringaepv.phaseolicola1448A

            HK type

            PSYR0492

            PSPTO5030

            PSPPH0483

            CheA-like

            PSYR1292

            PSPTO1482

            PSPPH1362a

            IB

            PSYR1300

            PSPTO1490

            PSPPH1371

            IC

            PSYR1307

            PSPTO1497

            PSPPH3877

            CheA-like

            PSYR1585

            PSPTO3900

            PSPPH1568

            IB

            PSYR1778

            PSPTO3696

            PSPPH1729

            IC

            PSYR1939

            PSPTO2129

            PSPPH1905

            IB

            PSYR2021

            PSPTO2212

            PSPPH1991a

            IB

            PSYR2113

            PSPTO2326a

            PSPPH2083a

            IB

            PSYR2445

            PSPTO2712

            PSPPH2601

            IB

            PSYR2448

            PSPTO2715

            PSPPH2604

            IB

            PSYR2450

            PSPTO2717

            PSPPH2606

            IC

            PSYR2700

            PSPTO2896

            PSPPH2483

            IC

            PSYR2940

            --

            --

            IB

            PSYR3355

            PSPTO3584

            PSPPH3276

            IC

            PSYR3532

            PSPTO1870

            PSPPH3473

            IC

            PSYR3612

            PSPTO1782

            PSPPH3628

            IB

            PSYR3698/GacS

            PSPTO1691

            PSPPH3719

            IB

            PSYR3996

            PSPTO4293

            PSPPH4003

            IC

            PSYR4408

            PSPTO4868

            PSPPH4451

            IB

            --

            PSPTO0896

            PSPPH4242

            IB

            --

            PSPTO0898

            PSPPH0796

            IB

            --

            PSPTO4079

            --

            IB

            --

            --

            PSPPH0770

            IB

            --

            --

            PSPPH0944

            IC

            --

            --

            PSPPH1261

            IC

            aGenes with disrupted reading frames.

            HK and RR genes were scattered over the entire chromosomes of the threeP. syringaepathovars. Conservation of the genetic organization between HK and RR genes was analysed in the genomes ofPsyB728a,PtoDC3000 andPph1448A allowing the identification of gene clusters containing HKs and RRs that constitute putative TCSs (Table 2). Like in other bacterial species, manyP. syringaeHKs and RRs were encoded by clusters of adjacent genes: 37 putative clusters of complete TCS genes inPsyB728a, 34 inPtoDC3000, and 33 inPph1448A (Table 2). For the remaining HK or RR genes, their partner genes could not be predicted from genetic organization and, therefore, they were considered as orphan HKs or RRs. The orphan HKs were 11 in eachP. syringaegenome, and the number of genes encoding orphan RRs was very high: 36 inPsyB728a, 38 inPtoDC3000 and 35 inPph1448A (Table 3). Finally, the comparative genomic analysis allowed the identification of a core of complete TCS protein orthologues among the threeP. syringaepathovars, that is composed by 30 putative TCS clusters (HK and RR) (Table 2), 11 orphan HKs, 33 orphan RRs (Table 3), and 16 hybrid HKs (Table 4).
            Table 2

            Putative TCS gene clusters in the genomes ofP. syringaepv.syringaeB728a, pv.tomatoDC3000 and pv.phaseolicola1448A.

            Histidine kinase/Response regulator

            Protein namea

            Organizationb

            HK type

            RR type

            Psy B728a

            Pto DC3000

            Pph 1448A

                

            PSYR0064/0063

            PSPTO0126/0127

            PSPPH0070/0069

            FimS/AlgR

            HR

            LytS-like

            LytR-like

            PSYR0259/0258

            PSPTO0329/0328

            PSPPH0247/0246

            EnvZ/OmpR

            RH

            IA

            OmpR-like

            PSYR0264/0263

            PSPTO0335/0334

            PSPPH0253/0252

            --/AlgB

            RH

            IA

            NtrC-like

            PSYR0723/0722

            PSPTO0824/0823

            PSPPH0737/0736

            PilS/PilR

            RH

            IC

            NtrC-like

            PSYR0786/0788

            PSPTO0913/0915

            PSPPH0805/0807

            CheA1/CheY1

            RHc

            CheA-like

            Stand-alone REC

            PSYR0832/0831

            PSPTO0965/0964

            PSPPH0858/0857

            --/--

            HR

            IC

            NtrC-like

            PSYR1100/1099

            --/--

            PSPPH1168/1167

            --/--

            RHd

            IB

            PleD-like

            PSYR1112/1111

            PSPTO1291/1290

            PSPPH1180/1179

            --/GltR

            RH

            IA

            OmpR-like

            PSYR1126/1127

            PSPTO1306/1307

            PSPPH1194/1195

            --/--

            RH

            IA

            OmpR-like

            PSYR1498/1497

            --/--

            --/--

            CopS/CopR

            RH

            IA

            OmpR-like

            PSYR1941/1940

            PSPTO2131/2130

            PSPPH1907/1906

            --/--

            HR

            III

            NarL-like

            PSYR2031/2032

            PSPTO2222/2223

            PSPPH2003/2004

            RhpS/RhpR

            RH

            IA

            OmpR-like

            PSYR2050/2051

            PSPTO2245/2246

            PSPPH2021/2022

            KdpD/KdpE

            HR

            IA

            OmpR-like

            PSYR2374/2375

            PSPTO2642/2643

            PSPPH2510/--e

            --/--

            RH

            IA

            OmpR-like

            PSYR2385/2384

            PSPTO2652/2651

            --/--

            BphP2/--

            HR

            GAF-HK

            Stand-alone REC

            PSYR2867/2868

            PSPTO2983/--e

            PSPPH2377/2376

            BaeS2/BaeS1

            HR

            IA

            OmpR-like

            PSYR3085/3084

            --/--

            PSPPH2980/--e

            --/--

            RH

            IA

            OmpR-like

            PSYR3128/3127

            PSPTO3298/3297

            PSPPH3041/3040

            --/--

            RH

            IA

            OmpR-like

            PSYR3211/3212

            PSPTO3380/3381

            PSPPH3126/3127

            --/--

            RH

            IA

            OmpR-like

            PSYR3375/3374

            PSPTO3604/3603

            PSPPH3295/3294

            --/--

            RH

            IA

            OmpR-like

            PSYR3434/3436

            PSPTO1982/1980

            PSPPH3360/3362

            CheA2/CheY2

            RHc

            CheA-like

            Stand-alone REC

            PSYR3460/3459

            PSPTO1955/1956

            PSPPH3386/3385

            FleS/FleR

            HR

            IC

            NtrC-like

            PSYR3512/3511

            PSPTO1893/1894

            PSPPH3454/3453

            QseC/QseB

            RH

            IA

            OmpR-like

            PSYR3708/3709

            PSPTO1680/1679

            PSPPH3729/3730

            PhoQ/PhoP

            RH

            IA

            OmpR-like

            PSYR3715/3716

            PSPTO1673/1672

            PSPPH3736/3737

            --/RstA

            RH

            IA

            OmpR-like

            PSYR3792/3793

            --/--

            PSPPH1461/1460

            --/CpxR

            RH

            IA

            OmpR-like

            PSYR3912/3913

            PSPTO4175/4176

            PSPPH3906/3907

            --/--

            HR

            IC

            NtrC-like

            PSYR3964/3965

            PSPTO4230/4231

            PSPPH3961/3962

            TctE/TctD

            RH

            IA

            OmpR-like

            PSYR3994/3995

            PSPTO4291/4292

            PSPPH4001/4002

            --/--

            HR

            IC

            NtrC-like

            PSYR4069/4070

            PSPTO4373/4374

            PSPPH4074/4075

            ColS/ColR

            RH

            IA

            OmpR-like

            PSYR4231/4230

            PSPTO4554/4553

            PSPPH4256/4255

            --/--

            HR

            IC

            PrrA-like

            PSYR4619/4618

            PSPTO0559/0560

            PSPPH0641/0642

            --/--

            HR

            III

            NarL-like

            PSYR4799/4800

            PSPTO0379/0378

            PSPPH4827/4828

            --/--

            RH

            IA

            OmpR-like

            PSYR4821/4822

            PSPTO0353/0352

            PSPPH4852/4853

            NtrB/NtrC

            HR

            IC

            NtrC-like

            PSYR4937/4938

            PSPTO5398/5399

            PSPPH0147/0146

            --/--

            HR

            IC

            NtrC-like

            PSYR5033/5032

            PSPTO5478/5477

            PSPPH5115/5114

            PhoR/PhoB

            RH

            IA

            OmpR-like

            PSYR5089/5088

            PSPTO5549/5548

            PSPPH5172/5171

            --/--

            HR

            IC

            LytR-like

            --/--

            PSPTO0785/0786

            --/--

            --/--

            HR

            IA

            OmpR-like

            --/--

            PSPTO4705/4704

            --/--

            CorS/CorR

            RH

            IB

            NarL-like

            --/--

            PSPTO5573/5574e

            --/--

            --/--

            HR

            IC

            OmpR-like

            aWhenever a HK or RR ofP. syringaehas been assigned a function in the literature and/or an annotation in databases, the corresponding protein name is mentioned;borganization of each TCS onP. syringaegenomes (HR, 5' histidine kinase-3' response regulator; RH, 5' response regulator-3' histidine kinase);can additional gene is located in between the RR and HK genes;dHR inP. syringaepv.phaseolicola1448A;egenes with disrupted reading frames.

            Table 3

            Orphan HK and RR genes in the genomes ofP. syringaepv.syringaeB728a, pv.tomatoDC3000 and pv.phaseolicola1448A.

            P. syringaepv.syringaeB728a

            P. syringaepv. tomato DC3000

            P. syringaepv.phaseolicola1448A

            HK/RR type

            Orphan HKs

               

            PSYR1918

            PSPTO2123

            PSPPH1874

            GAF-HK

            PSYR2978

            PSPTO3111

            PSPPH2262

            IC

            PSYR3060

            PSPTO3195

            PSPPH2185

            IC

            PSYR3504/BphP1

            PSPTO1902

            PSPPH3446

            GAF-HK

            PSYR3591

            PSPTO1803

            PSPPH3550

            IA

            PSYR3773

            PSPTO1606

            PSPPH1480

            GAF-HK

            PSYR3774

            PSPTO1605

            PSPPH1479

            GAF-HK

            PSYR4089

            PSPTO4395

            PSPPH4095

            IC

            PSYR4339

            PSPTO4796

            PSPPH4381

            IB

            PSYR4373

            PSPTO4833

            PSPPH4416

            IC

            PSYR4439

            PSPTO4896

            PSPPH4481

            GAF-HK

            Orphan RRs

               

            PSYR0089

            PSPTO0303

            PSPPH0094

            Stand-alone REC

            PSYR0488/PilG

            PSPTO5034

            PSPPH0479

            Stand-alone REC

            PSYR0489/PilH

            PSPTO5033

            PSPPH0480

            Stand-alone REC

            PSYR0509

            PSPTO5014

            PSPPH0499

            PleD-like

            PSYR0781/CheB1

            PSPTO0908

            PSPPH0800

            CheB-like

            PSYR0886

            PSPTO1039

            PSPPH0923

            CheC-like

            PSYR1098

            PSPTO1278

            PSPPH1166

            PleD-like

            PSYR1139

            PSPTO1323

            PSPPH1207

            CheW-like

            PSYR1190/HrpR

            PSPTO1379

            PSPPH1270

            NtrC-like

            PSYR1191/HrpS

            PSPTO1380

            PSPPH1271

            NtrC-like

            PSYR1293

            PSPTO1483

            PSPPH1363

            VieA-like

            PSYR1294

            PSPTO1484

            PSPPH1364

            NarL-like

            PSYR1308/CheB2

            PSPTO1498

            PSPPH3876

            CheB-like

            PSYR1309/WspR

            PSPTO1499

            PSPPH3875

            PleD-like

            PSYR1384

            PSPTO4027

            PSPPH3800

            NarL-like

            PSYR1912

            PSPTO2117

            PSPPH1867

            RsbU-like

            PSYR1938

            PSPTO2128

            PSPPH1904

            Stand-alone REC

            PSYR2114

            --

            --

            NarL-like

            PSYR2115

            PSPTO2330

            --

            Stand-alone REC

            PSYR2449

            PSPTO2716

            PSPPH2605

            Stand-alone REC

            PSYR2897/GacA

            PSPTO3024

            PSPPH2328

            NarL-like

            PSYR2939

            --

            --

            AmiR-like

            PSYR3091

            PSPTO3245

            PSPPH2995

            OmpR-like

            PSYR3299

            PSPTO3526

            PSPPH3220

            NarL-like

            PSYR3433/CheB3

            PSPTO1983

            PSPPH3359

            CheB-like

            PSYR3451

            PSPTO1964

            PSPPH3377

            RsbU-like

            PSYR3461/FleQ

            PSPTO1954

            PSPPH3387

            NtrC-like

            PSYR3486

            PSPTO1927

            PSPPH3413

            CheW-like

            PSYR3496

            PSPTO1911

            PSPPH3428

            VieB-like

            PSYR3589

            PSPTO1806

            PSPPH3547

            OmpR-like

            PSYR3890

            PSPTO4151

            PSPPH1374

            NarL-like

            PSYR4376

            PSPTO4836

            PSPPH4419

            NarL-like

            PSYR4377

            PSPTO4837

            PSPPH4420

            PleD-like

            PSYR4388

            PSPTO4848

            PSPPH4431

            Stand-alone REC

            PSYR4701

            PSPTO0472

            PSPPH4737

            Stand-alone REC

            PSYR5036

            PSPTO5482

            PSPPH5118

            Stand-alone REC

            --

            PSPTO0897

            PSPPH4241

            NarL-like

            --

            PSPTO2329a

            --

            Stand-alone REC

            --

            PSPTO4080

            --

            NarL-like

            --

            PSPTO4706/CorP

            --

            NarL-like

            --

            --

            PSPPH0778

            NarL-like

            aGenes with disrupted reading frames.

            Classification of HKs

            HKs have been classified on the basis of phylogenetic analyses and the sequence relationships of the residues surrounding the H-box [7,8,17,34]. Furthermore, several new domains with putative biological functions have been described in HKs, and domain architecture has proven particularly informative for analysing multi-domain proteins involved in signal transduction [2,11,12,35]. The phylogenetic analysis and examination of the region around the H box ofP. syringaeHKs showed that three of the five major HK types found inE. coli[8] were present inP. syringae: Type I (IA, IB, IC), III, and CheA-like HKs (Table 1; see Additional File1). In contrast, Type II and IV HKs were totally absent fromP. syringae. However, the LytS-like HK FimS/AlgZ and HKs containing GAF domains did not cluster within any of the defined HK types ofE. coli[8], and formed two separate HK groups: LytS-like HKs and GAF-HKs. GAF sensor domains are commonly found cytoplasmic signalling domains in the N-terminal region of HKs [2,34], and appear to act as binding sites for small ligands, such as cyclic nucleotides (cAMP and cGMP) and small molecules, which modulate the catalytic activity of the target protein [36,37]. In addition, analysis of domain architecture ofP. syringaeHKs showed a conserved core structure for each HK type inP. syringae(Figure 1). The conserved core of Type III HKs and LytS-like HKs only had a HK-like ATPase (HATPase_c) catalytic domain and a His_kinase domain, respectively. The conserved core of CheA-like HKs contained a C-terminal CheA regulatory domain but lacked the HisKA domain. The conserved core of Type I HKs and GAF-HKs had a central region with HisKA and HATPase_c domains fused to additional domains on the N-terminal end: a HAMP domain in Type IA, a PAS domain in Type IC, and GAF plus phytochrome (PHY) binding domains in GAF-HKs (Figure 1).
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-397/MediaObjects/12864_2007_Article_1110_Fig1_HTML.jpg
            Figure 1

            Schematic representation of the conserved core structures found inP. syringaeHK types. The domains are not drawn to scale. HAMP, domain found inHKs,Adenylyl cyclases,Methyl binding proteins andPhosphatases (PF00672); HisKA, HK dimerization/phosphoaceptor domain (PF00512); HATPase_c, HK-type ATPase catalytic domain (PF02518); REC, receiver domain (PF00072); PAS, signal sensor domain (PF00989); HPt,Histidine-containingPhosphotransfer domain (PF01627); H-kinase_dim, HK homodimeric domain (PF02895); GAF, signal sensor domain (PF01590); PHY, phytochrome domain (PF00360); His_kinase, region within bacterial HKs (PF06850).

            Orphan HKs fell into two HK types: Type I (IA, IB and IC), and GAF-HKs (Table 3); and hybrid HKs ofP. syringaebelong either to the Type I (IB and IC) or CheA-like HKs (Table 4). PSYR3504 (BphP1) and PSYR2385 (BphP2) HKs have been previously described as bacteriophytochromes (BphPs) that belong to the HWE_HK family [4,38]. Similar to other BphPs, thebphP1(PSYR3504) gene ofP. syringaepathovars is located in an operon downstream from abphOgene, encoding a putative heme oxigenase.

            Classification of RRs

            RRs show a great variety of output domains and domain combinations. Recently, bacterial and archaeal RRs have been classified into families based in their domain architectures [12]. RRs typically consist of an N-terminal REC domain fused to a C-terminal HTH DNA-binding output domain (OmpR, NarL, NtrC, LytR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) that activates or represses transcription of specific target genes [2,12]. In addition, prokaryotic genomes encode a variety of RRs with unusual domain organization: RRs with enzymatic output domains (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisKA), RRs with RNA-binding output domains (ANTAR and CsrA), RRs with protein- or ligand-binding output domains (CheW, PAS, GAF, TPR, CAP_ED, and Hpt), RRs with the REC domain as a stand-alone module, and RRs with domains of unknown function [12]. The RRs identified from the genomes ofP. syringaepathovars were assigned to these different RR families [12] according to the domain architecture and phylogenetic analysis (Table 1; see Additional File2).

            Bacterial RRs without a REC domain are extremely rare, but a number of enhancer-binding proteins (EBPs) lack the REC domain and normally function as RRs [39]. EBPs are involved in the activation of the bacterial transcription by interaction with the sigma-54 RNA polymerase holoenzyme [40]. InP. syringae, the HrpR and HrpS proteins show a high sequence similarity to the NtrC family of transcriptional RRs and have been previously identified as unusual EBPs lacking the N-terminal REC domain; however, similar to other EBPs, they retain the domain that interacts with the sigma-54 RNA polymerase holoenzyme plus the C-terminal DNA-binding domain [3942]. In addition, the NarL-like RR CorP ofPtoDC3000 that is involved in the regulation of coronatine biosynthesis [43,44] also lacks the REC domain. Thus, HrpR, HrpS and CorP proteins were not identified during the search of RRs inP. syringaegenomes with the HMM profile that targets the RR REC domain, nevertheless these proteins were considered orphan RRs (Table 3).

            Differences in TCS genes among pathovars that may contribute to plant host specifiCity

            A close analysis of the distribution of genes encoding TCS proteins revealed that there are important differences in TCS proteins among the three pathovars ofP. syringaethat may contribute to their diverse host ranges and association with particular host plants. A number of the identified TCS genes were unique to eachP. syringaepathovar without counterparts in the other two pathovars. ThecorRSPregulatory region (PSPTO4704-4706) of coronatine biosynthesis and thecopRSTCS (PSYR1497/1498) regulating copper resistance were only present inPtoDC3000 andPsyB728a, respectively. Other TCS genes unique to eachP. syringaepathovar were: PSYR2114, PSYR2939, PSYR2940 and PSYR3084 inPsyB728a; PSPTO0785/0786, PSPTO2329, PSPTO4079, PSPTO4080 and PSPTO5573/5574 inPtoDC3000; PSPPH0770, PSPPH0778, PSPPH0944 and PSPPH1261 inPph1448A. The unique hybrid HKs PSPPH0770 and PSPPH0944 were flanked by transposases. However, the unique RRs PSPTO2329 and PSPTO5574 were disrupted by transposases [25,27], and it is unlikely that these genes encode functional products. Finally, 11 TCS proteins were only shared between two of theseP. syringaepathovars.

            Variations amongP. syringaepathovars were also produced by the insertion of mobile genetic elements or point mutations in TCS genes resulting in disrupted reading frames. PSPTO2326 and PSPPH2083 encoded truncated hybrid HKs by comparison with the length of their orthologue PSYR2113 (Table 4) that is located next to the unique RR PSYR2114. PSPTO2326 and PSPPH2083 were located adjacent to a transposase and to a site-specific recombinase, respectively. Probably these elements caused the disrupted hybrid HKs and the lack of PSYR2114 orthologues inPtoDC3000 andPph1448A. Similarly, PSPTO2983 (baeS2) and PSPPH2510 encoded truncated HKs compared to the length of theirP. syringaeorthologues, and PSPPH2980 was interrupted by an ISPsy18 transposase. PSPTO2983, PSPPH2510 and PSPPH2980 HKs were unpaired without a RR gene in its vicinity, whereas theirP. syringaeorthologues are located on TCS gene clusters with adjacent RRs (Table 2).

            Although the PSPPH1362 gene was disrupted by an authentic frameshift,PsyB728a (PSYR1292) andPtoDC3000 (PSPTO1482) orthologues encoded intact hybrid HKs with similarity to BvgS ofBordetellaspecies that controls the regulation of many virulence factors [45]. In each pathovar, these hybrid HK genes were adjacent to orphan RR genes transcribed in the same direction (PSYR1293, PSPTO1482 and PSPPH1363), and their encoded proteins exhibited significant homology to the PvrR RR ofP. aeruginosaPA14 which controls antibiotic susceptibility and biofilm formation [46], and to the virulence related protein VieA ofVibrio cholerae[47].

            Conclusion

            In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes ofP. syringae. A large set of TCS proteins is required for the capaCity ofP. syringaeto detect and adapt to changing environments during plant association and pathogenesis. Moreover,P. syringaehas been isolated from non-plant environments such as river epilithon (rock-attached biofilms) [48] in which TCS proteins may have also important regulatory roles.P. syringaepathovars posses between 68–70 HKs and 92–95 RRs (Table 1), however there is little information describing their regulatory functions and the major part of these TCS proteins is uncharacterized. Many of the TCS proteins investigated so far inP. syringaehave been shown to be involved in plant pathogeniCity and association with host plants. The orphan RRs HrpR and HrpS are involved in a complex regulatory cascade that activates the transcription of the Hrp type III secretion genes and all known effector genes [42,49]. Expression of the type III secretion genes and effector genes is also regulated by the particular TCS GacA/GacS [50] and the RhpRS system [51]. Furthermore, the GacA/GacS system controls the expression of a variety of virulence factors, including protease and syringomycin biosynthesis [52]. The TCS CopRS and the modified CorRSP system regulate resistance to copper [53] and coronatine synthesis [43,44], respectively. Finally, the hybrid HK PSPTO2896 contains an N-terminal LOV (light, oxygen, or voltage) domain and is blue-light-activated [54].

            Bacteria with large genomes are disproportionately enriched in regulatory proteins involved in transcription control and signal transduction compared to medium and small-size genomes, and typically have complex regulatory networks relative to bacteria with smaller genomes [5557]. The existence of large numbers of HKs and RRs inP. syringaestrongly suggests that TCS proteins play important regulatory roles in the adaptation of this bacterium to different plant and non-plant environments. Comparative genomics of closely related species of pathogenic bacteria represents a powerful tool for the identification of genes potentially involved in host specifiCity and pathogenesis. The availability of the genome sequences ofPtoDC3000,PsyB728a andPph1448A provides us with the unique capability of comparing the complement of TCS proteins in theseP. syringaepathovars that differ in host range and other interactions with plants. This comparative genomic analysis reveals a core of orthologues and important differences in TCS genes betweenP. syringaepathovars. It is especially worth noting the high number of genes encoding orphan HKs and RRs in these genomes. Moreover, differences in the repertoires of TCS proteins are likely to facilitate the adaptation ofP. syringaepathovars to different plant hosts and/or could be responsible for the different disease characteristics induced. Consequently, the TCS proteins unique to eachP. syringaepathovar are interesting targets for future investigations to identify TCS proteins involved in the different host ranges and/or plant pathogenesis. However, the challenge remains to associate these differences in TCS proteins to specific traits ofP. syringaepathovars. Additionally, pathovar-specific differences in gene content might be used to design targeted approaches for disease control and could allow the precise PCR-based diagnosis of bacterial diseases [58].

            Analysis of the regulatory functions, molecular mechanisms and signal transduction pathways of TCS proteins should contribute to the understanding of the complex events that occur inP. syringaeduring pathogenesis and adaptation to different plant hosts and different non-plant environments. Rapid progress in the study of TCS proteins is being made by the combination of molecular genetic approaches with genome-scale analysis [59]. Genetic and biochemical studies are necessary to further explore the signal transduction pathways mediated by some of these TCS proteins at the molecular level: construction and analysis of deletion mutants in TCS genes in order to determine the signals sensed by the HK and the targets for the RR of each system. In addition, the application of more extensive analysis with global methods, such as DNA microarray studies reported forB. subtilis[60] andS. pneumoniae[61], might allow defining the regulons and the potential regulatory functions of TCS proteins in response to environmental signals. Furthermore, unravelling these signal transduction pathways could potentially lead to the design of innovative strategies to controlP. syringae. In conclusion, this comparative genomic analysis constitutes a basis for future functional genomic analysis ofP. syringaeto establish which TCS proteins participate in the pathogenesis and the adaptation to different plant and non-plant environments.

            Methods

            Identification of TCS proteins inP. syringaegenomes

            The identification of HKs and RRs is based on the computational domain analysis of protein sequences. The approach used to identify putative HKs and RRs from the complete genome sequences ofPsyB728a,PtoDC3000 andPph1448A was similar to that described previously [33] with slight modification. Briefly, five different HMM profiles (accession numbers PF00512, PF07568, PF07730, PF07536 and PF06580) were found in Pfam database that target different families of HKs (HisKA, HisKA_2, HisKA_3, HWE_HK and His_kinase). The HWE_HK domain is defined by the absence of a recognizable F box, and the presence of a highly conserved H residue and a WxE motif within the N and G1 boxes of the C-terminal transmitter domain, respectively [4]. These five different HMM profiles were used to recognize the different HKs in theP. syringaegenomes, and hits with a E-value below a selected cut-off (10-6) were extracted. A profile HMM downloaded from Pfam protein families database [62], which targets the RR REC domain (accession number PF00072), was used to recognize the RRs in eachP. syringaegenome. Hits with an E-value below a selected cut-off (10-12) were extracted. Additionally, the CheA HK ofEscherichia coli[31] was used as template in BLASTP searches to identify CheA-like HKs in theP. syringaegenomes and hits with an E-value below a selected cut-off (10-10) were extracted. Hybrid HKs (REC-HKs) were determined by the presence of complete HK transmitter and REC domains in a single protein. Detection of orthologues of the identified HKs and RRs between the genomes ofPsyB728a,PtoDC3000 andPph1448A was determined by BLASTP [63] based on the reciprocal best hits of eachP. syringaegenome against each other genome, completed by the phylogenetic analyses. Finally, functional domains of the HKs and RRs were identified by search the Conserved Domain Databases (CDD) with Reverse Specific Position BLAST [64].

            Sequence alignment and phylogenetic analysis

            Multiple sequence alignments and phylogenetic trees of HKs and RRs were constructed using the ClustalW program [65], and aligned sequences were imported into the MEGA 3.1 program [66] where phylogenetic trees were inferred. Default parameters were used. Phylogenetic trees were subdivided into groups of orthologues, and co-clustering with members of specific TCS proteins allowed a definitive assignation to a given HK type or RR family.

            List of abbreviations

            TCS: 

            two-component system

            HK: 

            histidine kinase

            RR: 

            response regulator

            HMM: 

            Hidden Markov Model

            HTH: 

            helix-turn-helix

            ECF: 

            extracytoplasmic function

            EBP: 

            enhancer-binding protein

            Psy

            Pseudomonas syringaepv.syringae

            Pto

            P. syringaepv.tomato

            Pph

            P. syringaepv.phaseolicola

            REC: 

            receiver

            PHY: 

            phytochrome

            LOV: 

            light, oxygen, and voltage

            Declarations

            Acknowledgements

            JLL was a recipient of a predoctoral fellowship from the Public University of Navarra. JAO was supported by the Ramón y Cajal Programme and Complementary Action Grant BIO2006-28484-E of the Spanish Ministerio de Educación y Ciencia (MEC). JLL and JAO thank Antonio G Pisabarro and Lucía Ramírez for continued support. DWU and KK would like to acknowledge funding by the Danish Center for Scientific Computing.

            Authors’ Affiliations

            (1)
            Departamento de Producción Agraria, Universidad Pública de Navarra
            (2)
            Center for Biological Sequence Analysis, Biocentrum-DTU, The Technical University of Denmark

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            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.