Vol. 171, No. 9JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 5031-50380021-9193/89/095031-08$02.00/0

The Predicted Protein Product of a Pathogenicity Locus fromPseudomonas syringae pv. phaseolicola Is Homologous to a Highly

Conserved Domain of Several Procaryotic Regulatory ProteinsC. GRIMM"2 AND N. J. PANOPOULOS2*

Institute of Molecular Biology and Biotechnology, Research Center of Crete, Heraklio, Greece,' andDepartment ofPlant Pathology, University of California, Berkeley, California 947202

Received 1 December 1988/Accepted 22 May 1989

A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicolawith its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partiallycharacterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS),located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a codingcapacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product ofORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC,NifA, and DctD proteins ofRhizobium spp., the NtrC and NifA proteins ofKlebsiella pneumoniae, and the TyrRprotein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport andmetabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be theshortest naturally occurring member of this family of proteins, corresponding for the most part to the highlyconserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segmentanalogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present inHrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding thata functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacteriuminteraction.

The plant pathogen Pseudomonas syringae pv. phaseoli-cola causes the halo blight disease on bean, its natural host,and elicits the hypersensitive response (HR), a resistance-associated reaction, on several nonhost plants. In previousstudies, we described a group of genes that are required forpathogenicity as well as for the elicitation of HR by thisbacterium (21). We have designated these genes hrp (pho-netic "harp," for hypersensitive reaction and pathogenicity)and have established that in strain NPS3121, the majority ofthem are clustered in a 20- to 22-kilobase (kb) region, the hrpcluster. HR is an expression of plant resistance to heterolo-gous pathogens, but is also expressed in resistant cultivars ofhost species that carry resistance genes that are functionallycorrespondent in a gene-for-gene sense (10) to avirulencegenes (avr) present in the pathogen (39, 40). Significantly,the expression of race- or cultivar-specific HR requires afully functional hrp region, in addition to avirulence genes(20).The mechanism by which the hrp genes determine or

control the plant-pathogen interactions is not known. Togain further insight into the subject, detailed structural andfunctional analysis of hrp genes has been undertaken. In thisreport we present the DNA sequence, complementationanalysis, and in vitro expression of one hrp locus, which wedesignated hrpS. We show that this locus contains a largeopen reading frame (ORF) and that the predicted translationproduct of this ORF is a 302-amino-acid protein. This proteinshows significant homology to the highly conserved centraldomain of several procaryotic regulatory proteins that areinvolved in the activation or repression of diverse operons inenteric and plant symbiotic bacteria in response to different

* Corresponding author.

environmental or cellular signals. We also show that hrpS isa regulatory locus whose function is necessary for theexpression of another hrp gene during the host-pathogeninteraction. The short length of this protein compared withthe length of other members of the group corroboratesprevious predictions of functional autonomy of their highlyconserved domains (7) and invites speculation about theevolution of regulatory mechanisms important to Pseudomo-nas pathogenesis on plants.

MATERIALS AND METHODS

Enzymes and chemicals. The DNA-directed transcription-translation kit, Klenow polymerase, the random DNA label-ing kit, [35S]dATP, [32P]dCTP, and [35S]methionine wereobtained from Amersham Corp.; RNase-free DNase I andSP6 polymerase were from Promega Biotec; and the M1317-mer sequencing primer used was from B3ethesda ResearchLaboratories, Inc. Antibiotics were used as described inLindgren et al. (21).

Bacterial strains and vectors. Escherichia coli strainsNM522 [A(lac pro) supE recA(F' lacPqZAM15 pro')] andJM1l1 [A(lac proAB) supE thi rK- mK+(F' traD36 proABlacIqZAM15)] were used for subcloning purposes and forpropagation of M13 clones, respectively. NPS3121 is arifampin-resistant P. syringae pv. phaseolicola wild-typestrain (Hrp+, i.e., pathogenic on bean cultivar Red Kidneyand causing HR on tobacco cultivar Turk). NPS4006 is aHrp- Tn5 insertion mutant ofNPS3121 (21). NPS3121-12 (L.G. Rahme, M. N. Mindrinos, and N. J. Panopoulos, manu-script in preparation) and NPS4006-12 (M. N. Mindrinos, L.G. Rahme, and N. J. Panopoulos, manuscript in preparation)are marker exchange derivatives of NPS3121 and NPS4006,respectively, that have the same chromosomal insertion of

5031

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

5032 GRIMM AND PANOPOULOS

transposon Tn3-Spice (P. B. Lindgren, Ph.D. thesis, Univer-sity of California, 1987) in the hrpD locus (see Results).The broad-host-range vectors pLAFR3 (40) and pTE3 (8),

which were used for the complementation analysis, arederived from pRK290 (6). pLAFR3 contains the 425-base-pair 3-galactosidase (ox-peptide) HaeII fragment from pUC8cloned into the EcoRI site of cosmid pLAFR1 (11) afterfilling in with Klenow polymerase (40). This fragment alsoincludes the lac promoter and the pUC8 multilinker se-quence. pTE3 contains the Salmonella typhimurium trppromoter and the E. coli rpoC terminator on two separatefragments that were cloned into the HindlIl and EcoRI sitesof a multilinker identical to that in pUC8, respectively, suchthat the vector retained unique cloning sites for BamHI andPstI (8). ORF fusions to the C terminus of 3-galactosidasewere constructed with the pUR vector series (35). Theplasmids pSP64 and pSP65 (25) were used as Sp6 promotervectors for in vitro transcription.

Bacterial conjugations. Mobilization of recombinant plas-mids was done by triparental matings, with plasmidpRK2013 as a conjugative helper plasmid as describedpreviously (6).

Library and plasmids. The DNA inserts of plasmids usedin this work were derived from a cosmid clone, pCG42, andsubclones thereof, which originated from a P. syringae pv.phaseolicola library, PSP-Lib2 (Frederick and Panopoulos,manuscript in preparation). The library was constructed byligating partially digested (Sau3A), dephosphorylated, andsize-fractionated genomic DNA from wild-type strainNPS3121 into the BamHI-linearized pLAFR3 vector (40).The pCG42 cosmid was identified by probing PSP-Lib2 withthe 4.2-kb El-HI fragment (Fig. 1) derived from a previouslydescribed cosmid clone, pPL6 (20); colinearity with thecorresponding genomic region was verified by Southern blotanalysis (data not shown). The 4.9-kb EcoRI fragment (El-E2), the 5.7-kb KpnI fragment (K1-K2), and the 3.8-kbHindIII fragment (H1-H2) (Fig. 1) were initially subclonedinto pUC18 in both orientations in order to facilitate theconstruction of the plasmids described below.

(i) pCG4201 and pCG4202. Cosmid pCG42 was digestedwith HindIII, and the H1-H2 sites were religated. Theresulting plasmid was redigested with HindlIl, and theHindIII-EcoRI (H1-E2) fragment and the HindIII-KpnI (Hi-K2) fragment, isolated from the pUC18 subclones as HindIllfragments, were ligated into the HindlIl site to createpCG4201 and pCG4202, respectively. The orientation of theHindIII fragments in pCG4201 and pCG4202 was checked byrestriction site mapping.

(ii) pCG161 and pCG162. The 1.5-kb BglII-EcoRI (E2)fragment was isolated from a pUC18 subclone as a BglII-BamHI fragment and was ligated into the BamHI site ofpTE3 in both orientations.

(iii) pCG2784 and pCG2786. The 4.2-kb EcoRI-HindIII(El-Hi) fragment was isolated from a pUC18 subclone as aHindIII fragment and was ligated into the HindlIl site ofpUR278 in both orientations.

(iv) pCG654, pCG652, and pCG641. The 4.9-kb EcoRI(El-E2) fragment (pCG654) and the 4.2-kb EcoRI-HindIII(El-Hi) fragment (pCG641) were cloned into pSP65 andpSP64, respectively, such that hrpS transcription was underthe SP6 promoter control (see Fig. 4A). The EcoRI-HindIIIfragment (El-Hi) was also placed in the opposite orientationrelative to the SP6 promoter by cloning in pSP65 (pCG652).

(v) pCG45.21. The HindlIl fragment Hl-H2 was clonedinto the synonymous site of a modified pLAFR3 derivative,in which the EcoRI site was destroyed by a fill-in reaction

with Klenow polymerase, to produce a recombinant plasmidthat had the lac promoter distal from the HindlIl site H2 ofthe insert. This plasmid was then digested with EcoRI, andthe promoterless inaZ gene, isolated from plasmid Tn3-Spice(19a) as a 3.6-kb EcoRI fragment, was cloned into the EcoRIsite E2, so that inaZ and hrpS reading frames were orientedsimilarly (from right to left; Fig. 1).

(vi) pCG8. The promoterless inaZ gene was isolated fromplasmid pTn3-Spice (19a) as a 5.9-kb BamHI fragment andwas cloned into the BamHI site of pTE3, such that inaZexpression was under control of the trp promoter of pTE3.

(vii) pLAFR6::inaZ. Plasmid pLAFR6 carries the pUC18multilinker sequence inserted in the EcoRI site of pLAFR1(11) and flanked by synthetic trp terminators (D. Dahlbeckand B. J. Staskawicz, personal communication). PlasmidpLAFR6::inaZ carries inaZ as a 3.6-kb EcoRIIBamHI frag-ment isolated from pTn3-Spice (19a) and directionally clonedbetween the synonymous multilinker sites ofpLAFR6 (T. V.Huynh, D. Dahlbeck, and B. J. Staskawicz, submitted forpublication).

Plant inoculations. Hypersensitivity tests were routinelyperformed on tobacco leaves (cv. Turk) by injecting bacte-rial suspensions at 108 to 109 CFU/ml as previously de-scribed (21). Pathogenicity tests were carried out by inocu-lating primary leaves of 12-day-old bean (cv. Red Kidney)with bacterial suspensions containing 105 to 106 CFU/ml, asdescribed previously (21). In planta induction experimentswere also carried out in Red Kidney bean leaves as de-scribed elsewhere (19a). Ice nucleation activity of bacteria inplanta and in culture was measured at -9°C by a dropletfreezing technique as described in reference 19a. Data wereexpressed as ice nuclei per cell.

In vitro transcription and translation. Runoff transcriptionfrom the SP6 promoter was carried out in a reaction mixturecontaining 1 ,ug of plasmid DNA, 20 U of RNasin, 0.4 mMribonucleoside triphosphates, 10 U of SP6 RNA polymerase,10 mM dithiothreitol, 40 mM Tris hydrochloride (pH 7.5), 6mM MgCl2, 2 mM spermidine, and 10 mM NaCl. Themixture was incubated for 1 h at 40°C. A 1-U portion ofRNase-free DNase I was added, and the mixture was incu-bated for 15 min at 37°C. After phenol extraction, the RNAwas precipitated with 3 volumes of EtOH at -80°C for atleast 4 h. About 1 Lg of RNA was used for in vitrotranslation with the E. coli cell-free transcription-translationsystem. The proteins were labeled with [35S]methionine (10pCi) and were separated on a 10% polyacrylamide-sodiumdodecyl sulfate gel. After fixation in 10% acetic acid, the gelwas treated with the autoradiography enhancer Enlightningfor 30 min and dried, and the [35S]labeled products werevisualized by autoradiography.

Analysis of fusion proteins. E. coli cells carrying pURplasmid derivatives were grown to mid-log phase beforeinduction of lacZ expression by isopropyl-,3-D-thiogalacto-pyranoside (0.1 mM) and were subsequently grown foranother 2 h at 37°C. Cells were then suspended in 1 xLaemmli buffer (19) and incubated at 85°C for 10 to 15 minfor lysis. Total cellular proteins were analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and pro-teins were stained with Coomassie brilliant blue.DNA sequencing. For sequencing, the dideoxy-chain ter-

mination method (36) was used with the M13 system. TheDNA segments covering the region of interest were sub-cloned into M13 vectors mpl8 and mpl9. Both strands were

sequenced. Deletions were created either by using appropri-ate restriction enzymes or by using the rapid sequencing

J. BACTERIOL.

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

LOCUS HOMOLOGY OF P. SYRINGAE AND OTHER PROCARYOTES 5033

El K1

,[ pCG 42r pCG 4202

r pCG4201

pCG 162

pCG161

4006

Bg P Hi E2I h *

hrpS

U

* H

Bg 0 5fBg

--PI 0

FIG. 1. The top line shows a physical map of the right part of the hrp cluster of P. syringae pv. phaseolicola. The lines below show theplasmids used for complementation tests and are explained in Materials and Methods. The inserts of pCG42, pCG4201, and pCG4202 arecloned in pLAFR3; the inserts of pCG161 and pCG162 are cloned in pTE3. The chromosomal mutation in hrpS (mutant NPS4006) is a TnSinsertion and is shown as a triangle on the top. Closed and open squares indicate complementation and no complementation, respectively,of the Hrp- phenotype of mutant NPS4006 by each plasmid. The single arrow below the restriction map corresponds to the ORF found inthe hrpS locus. Filled arrows indicate the location and orientation of the trp promoter in pTE3. Restriction sites written in italic letters belongto vector multilinker sequences. P, PvuII; E, EcoRI; K, Kpnl; X; Bg, BglII; H, Hindlll; B, BamHI.

system described by Dale et al. (5). [35S]dATP was used forlabeling.

RESULTS

Analysis of the hrpS locus. The phenotype of mutantNPS4006 has been previously described in detail (21). Unlikeits wild-type parent, NPS3121, the mutant fails to elicit HRand is not pathogenic on bean. The mutant carries a singlecopy of Tn5 inserted in the right part of the hrp cluster (about0.3 kb to the left of the HindIll site H1), within a gene whichwe here designate hrpS. NPS4006 could be complemented toHrp+ upon introduction of plasmid pCG4202, but not ofplasmid pCG4201. This indicated that hrpS probably lies tothe left of the EcoRI site E2 (Fig. 1), but regulatory se-

quences necessary for hrpS expression may extend beyondthis site to the right. Two other plasmids were constructed inorder to locate the left border of hrpS. Plasmids pCG161 andpCG162 contain the 1.5-kb BglII-EcoRI (E2) fragment inopposite orientations in the broad-host-range vector pTE3,which carries the E. coli trp promoter (8). Plasmid pCG162complemented NPS4006, while pCG161 failed to do so. Thesimplest interpretation of the results obtained with the abovefour plasmids is that the 1.5-kb BglII-EcoRI fragment con-tains most or all of the hrpS coding region, but regulatoryelements required in cis for hrpS expression are locatedbetween the EcoRI site E2 and the KpnI site K2 (Fig. 1),assuming that the locus is transcribed from right to left (seebelow).DNA sequence analysis. The complementation data above

have indicated that the coding region of hrpS is locatedbetween the BglII site and the EcoRI site E2. Accordingly,the nucleotide sequence of this region was determined (Fig.2). Two potential coding regions were identified. The longestORF that starts with an ATG codon has a potential codingcapacity for a polypeptide of 302 amino acid residues(ORF302). It starts 25 nucleotides to the left of the first basein the HindlIl (Hi) recognition sequence and terminateswith a TGA codon adjacent to the BglII site (Fig. 2). Itsorientation is compatible with the results of our complemen-tation analysis. Computer analysis of the sequence databased on the Positional Base Preference method (38) showedthat ORF302 has a high probability of being a protein-codingregion, and this probability drops abruptly around theHindIII site H1 (data not shown). Furthermore, all potentialinitiation codons further upstream (up to about 540 nucleo-

tides) are followed by one or more stop codons in all threereading frames. The second potential coding region is lo-cated upstream of ORF302, in a different frame that termi-nates with the TAG codon at position 125 to 127 of thesequence shown in Fig. 2 and presumably extends upstreambeyond the EcoRI site E2.As discussed in the previous section, hrpS expression

appears to be dependent upon upstream regulatory elementslocated to the right of the EcoRI site E2. The incompleteORF which precedes hrpS appears to be the 3'-terminal endof an adjacent locus that may be jointly transcribed withhrpS (C. Grimm, D. Dahlbeck, N. J. Panopoulos, and B. J.Staskawicz, manuscript in preparation). The absence ofsequences that match the consensus -35/-10 hexanucle-otides of canonical promoters recognized by the major formof the E. coli polymerase (@70, RpoD) from the regionimmediately upstream of ORF302 is consistent with thisinterpretation. However, the region upstream of ORF302contains a sequence motif that resembles the -24/-12regions of promoters that are recognized by the minor formof E. coli RNA polymerase ((X54, RpoN) (13, 15, 32). This

1 a:A CTCTTCGA=lACC CT

60 TTCAGGAGTGACTCGAGACTETACCACCATGAAO A

119 GCT GCAGCAAC CTG TCC C CCOTCATCTO178 ATC CAT CTT CAT CAG CGC mT? CAT GAC GAC CTA GAC GAG GC CGT223 GTE CCA AAT CTC COG ATC GTC GCT CM AOC ATT TCG CM CTC COCT268 ATC CAT CTC TTG CTC TCA 0CC GAG ACC GCG ACT 0CC MA CAC ACT313 ATT CCC CAG COC ATT CAC ACA ATA TCT 0CC COT MA GCT CCC CTC358 TrGTCC ATC MT TGC GCT 0G0 ATT COG GAO TCG CTC GCA GAG ACT403 GAG CTC mTT OGA CTG CTC AGC CCT GCG TAC ACC OCT OCT GAC CCC448 TCC AGA CTC COC TAC ATC GM 0CCG CA CM OGC CGA ACC CTC TAT493 CTC CAT GAG ATC CAC AGC ATC CC CTC AGCC CTO CAO CCC MGCTC538 CTC ACG CTC CTC CM ACC CGA GCA CTC GM CCA CTC OCT TCG ACA583 TCG ACC ATC MC CTC CAT CTC TCC CTC ATC OCT TOG CCC CAG TCG628 TCT CTC CAT CAT 0CC CTC GAG CGA GCG MA TTE CGT COG CAT CTC673 TAC TmE CCC GTE MCSGOC TTC ACA CTC CAG CTC CCA CCC TEC CCC718 ACA CAG CCT GAG CCC ATT CTC CCC TTC TTC MG CGC TTE ATC GCC763 GCA GCT CCC MA GM CTG MC CTC CCC TCT CCC GAT CTC TGT CCC828 CTC CTT CAG CAG CTG CTC TTG GCG CAT GAG TOO CCC CCC MT ATC873 CCT CAG CTC MC CCT GCT GCA MG CCC CAT CTA TTC CCT TTC CCG918 OTO TEC GCT CTC GAC CCC CAG ACT GM GAA CAC TTG CCC TGT CGG963 CTC AM TCC CAG CTC AGO CG ATE GM AC CCCTO AT MGCAA G

1008 TCA CTC MGC CGCCAG COT MC TGC ATT GAT CCC GCA ACC CTG GAG1053 CTC GAC ATG CCA CGC CGC ACC CTC TAT CGA CGT ATC MG GAG TTG1098 CAG ATC TGATTTTGCACACCTGAA

FIG. 2. Nucleotide sequence of the hrpS region. A potentialShine-Dalgarno sequence (37) and putative -24 to -12 promotersequences are overlined. Sequence palindromes upstream ofORF302 are underlined. The HindlIl (Hi) and BgIII restriction sitesoccur at position 153 to 158 and 1099 to 1104, respectively. The stopcodon TAG of the putative upstream ORF is underlined at position125 to 127.

K2 H2

Hf

I kb

VOL. 171, 1989

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

5034 GRIMM AND PANOPOU LOS

FIG. 3. Proteins from a crude cell lysate of E. c oli NM522containing the plasmids pCG2784 (lanes 1 and 2) and pCG2786 (lanes3 and 4) were separated on a 9% sodium dodecyl sulfate-polyacryl-amide gel. Proteins were stained with Coomassie blue R-250. Themolecular masses of the protein standards are indicated at the left (inkilodaltons). Lanes 1 and 3 show the protein pattern of cells whichhad been induced with isopropyl-3-D-thiogalactopyranoside. Lanes2 and 4 show the protein pattern of cells which have grown withoutisopropyl-p-D-thiogalactopyranoside.

motif shows the 10-base-pair spacing between dinucleotidesthat is considered critical (GG in the -24 region and GC inthe -12 region; see reference 18). Furthermore, sequenceswith dyad symmetry that resemble rho-independent termi-nators (one is underlined in Fig. 2) and several other directand inverted repeats occur upstream of ORF302. Finally,sequences very similar (five of six nucleotides) to the puta-tive ribosome-binding sites of several procaryotic and phagegenes cataloged by Gold et al. (12) precede the putative ATGof ORF302. The functional significance of these featuresremains to be determined.

lacZ-hrpS fusion. In order to confirm that ORF302 is in factan ORF, we used all three plasmids of the pUR vector series,pUR278, 288, and 289 (35), to construct a series of plasmidsin which the HindIll site (Hi) upstream from ORF302 wasfused to the 3'-terminal end of the lacZ gene in all threeframes. For this purpose, the 4.2-kb HindIII-EcoRI (HI-El)fragment was isolated as a HindIll fragment from a pUC18subclone of the 4.9-kb EcoRI (El-E2) fragment and clonedinto the three pUR vectors in both orientations, respec-tively. As expected from the nucleotide sequence, onlyplasmid pCG2784 produced a fusion protein (Fig. 3). Thisprotein had an apparent molecular mass of about 150 kilo-daltons (kDa), indicating that an uninterrupted reading framecapable of coding for a polypeptide of about 34 kDa had beenfused to P-galactosidase (116 kDa). This almost exactlyequals the size of the ORF302 translation product, calculatedfrom its predicted amino acid composition to be 33.5 kDa.Neither pCG2786, which carries the Hl-EB insert in theopposite orientation in pUR278 (Fig. 3), nor any of the fourpUR288 and pUR289 derivatives carrying the same insert inboth possible orientations produced 3-galactosidase fusionproteins (data not shown).

In vitro transcription-translation of hrpS. To investigatethe in vitro translation product of hrpS, we subcloned theEcoRI fragment El-E2 (pCG654) and the EtcoRI-HindIIIfragment (El-HI) (pCG641) into the riboprobe vectorspSP65 and pSP64, respectively (Fig. 4A). The orientation

IMAa

FIG. 4. (A) Plasmids used for in vitro transcription-translation.The filled arrows indicate the location and orientation of the SP6promoter. The thin arrows indicate the position at which theplasmids have been linearized. (B) The proteins translated in vitrofrom the RNAs produced from the following plasmids: (i) pCG641,linearized with EcoRI; (ii) pCG654, linearized with BgII; and (iii) noRNA. The proteins were labeled with [35S]methionine and analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Themolecular masses of the protein standards are given at the left inkilodaltons.

was such that hrpS transcription was under the control of theSP6 promoter and, in the case of the EcoRI-HindIII frag-ment El-HI, also in the opposite orientation in pSP65(pCG652, not shown). The plasmids were linearized at theEcoRI site El (pCG641) far downstream of hrpS, at theHindIII site Hi (pCG652) immediately upstream or at theBglII site (pCG654) which overlaps the termination codon ofORF302, so that the runoff transcripts would represent theentire inserts or only to hrpS sequences. These transcriptsserved as templates in a coupled E. coli transcription-translation system. The in vitro translation products derivedfrom plasmids pCG654 and pCG641 had one common bandof approximately 35 kDa (Fig. 4B). This value is in closeagreement with the predicted size of the translation productof ORF302 and indicates that a translational start codon anda functional ribosome binding site (37) occur close to anddownstream from the Hindlll site Hi, as predicted from thenucleotide sequence. A second band of about 27 kDa pro-duced from plasmid pCG641 is probably translated down-stream from the hrpS locus, since it was not seen withplasmid pCG654, which was linearized at the predictedtranslational stop codon of ORF302. This band probablyrepresents the product of another hrp locus downstreamfrom hrpS. No translation product was obtained when theEcoRI-HindIII fragment El-Hi was inserted into pSP65 inthe opposite orientation (pCG652) (data not shown).The predicted ORF302 protein (HrpS) shares homology

with several regulatory proteins. The NBRF-PIR proteindatabase (version 16) was searched by using the FASTPprogram (22), in order to identify possible similarities toother proteins and thus obtain clues about the possible

J. BACTERIOL.

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

LOCUS HOMOLOGY OF P. SYRINGAE AND OTHER PROCARYOTES 5035

Kp NifA

Psp HrpS

Kp NtrC

Kp NifA

Psp HrpS

Kp NtrC

Kp NifA

Psp HrpS

Kp NtrC

IDI-TPSRGFGLENNVGKSPAMRQIMDIIRQVSRWDTRVLVRGESSTGULIM 251

MDLDEGFDDDLDEERVPNLOIVAESISQL---GIDVLLSGETGTGTIAQ 48[D)

- INSPTADIIGEAPAMQDVFRIIGRLSRS - --SISVLINGESCTGKELVAH 179

AIHHNSPRAAAAFVKFNCAALPDNLLESLFQHEKGAFrGAVRQRKGRFEL 302

RIHTISGR-KGRLVAXNCAAIPESLAESELFCVVSGAYTGADBSRVGYIIA 98

ALHRHSPRAKAPFIA1KAIPKDLIESELFCUUGAFGANTVRQG3F3Q 230

ADGGTLFLDEIGEISSASFQAKUJRIQEGIITVOGDKTLRVNIIVIMN 353

AQGGTLYLDEIDSNPLSLQAKlVLETRA STSTIKLDVCVIASAQ 149

ADGGTLFLDIGDEIITQTRll2V8ADOQFY1VGGYAVDVIIAATH281

Kp NifA RHLEK LOHFREDLY VPlWIALPPIZUQDIL V-AIFLV M 403

Psp HrpS SSLDDAVUQGKFRRDLYFRIJWLTLQLlIRTQPUILPFKR1KAAAAKI 200

Kp NtrC QNLKI.VQIGKfDFHDLVVl?IDIDIIAA 332

Is)Kp NifA SQGRTLRISDGAIRLUYSWP W VL LI 4

Psp HrpS UNASADVCPLLAVVLLOHEWPGIEF&VC?1LG IS 251

Kp NtrC ILCVKAQUHP TVRQlUIT QlTIITCGILTQDL? 363

Kp NifA

Psp HrpS

Kp NtrC

NHRDPPKALASSGPAEDGvLDNSLDRIQRLAA ilv4MAALLm- 505

IACGLKSQLBAIEKALIQQSIURHRN IDALS * 32*. @...... r[E]

ELFETAIpDNPTQKsPDwVATLLQWADRWZsGHQKLISKPIUTLL- 434

FIG. 5. Amino acid sequence alignment of the predicted proteinproduct of ORF302 and the regulatory proteins NtrC and NifA of K.pneumoniae. Double dots indicate identities, and single dots indi-cate conservative amino acid replacements. The identities were

43.2% between HrpS and KpNtrC and 42.8% between HrpS andKpNifA. Amino acids are given in the standard single letter code. Dand E indicate the starting points for domains D and E, respectively,of NifA and NtrC (7). The putative ATP-binding site is overlined;the putative helix-turn-helix motif is underlined.

functions of the predicted ORF302 product, which we des-ignate HrpS. Four bacterial proteins showed significantsimilarity to HrpS: NifA and NtrC of Klebsiella pneumoniae(KpNifA and KpNtrC), NifA of Rhizobium meliloti(RmNifA) (3), and DctD of Rhizobium leguminosarum(RlDctD) (31). The identities exceeded 40% in all cases. InFig. 5, the predicted amino acid sequence of HrpS has beenaligned with the amino acid sequence of KpNtrC andKpNifA. Only minor gaps were required to allow maximumalignment between the three proteins. Comparable levels ofsimilarity were also found between the HrpS product andother NtrC and NifA proteins (16, 18, 26, 28, 41) and with theTyrR protein of E. coli (4, 14). The above similarities wereconfined to the central, ca. 200-amino-acid region of HrpSand to the central regions of NtrC, NifA, DctD, and theC-terminal half ofTyrR (Fig. 6). The highly conserved regionamong these proteins corresponds to domain D of NifA andNtrC, as defined by Drummond et al. (7) (Fig. 6). There wasno significant similarity between the N- and C-terminalregions of HrpS and the positionally correspondent aminoacids of any of these proteins.

All known members of the NtrC protein family possessextended N-terminal domains that are thought to mediate theinteraction between these proteins and sensor proteins spe-cific to the particular signal transduction pathway in whicheach sensor-regulator pair operates (28, 43). Our in vitro

Ec TyrR

Psp HrpS

Rm NIfA

Kp NIfA

Kp NtrCI IB

..V- l l

DI I~~~~~~~~~~~~~~~~~~~1-472

E _1-302--- E 1-541

1-524

1-469* _,,0AfRI DctD - 1-448

At Vir6 _ 1-267

FIG. 6. Domain relationships of six regulatory proteins with theHrpS protein. The domains of KpNtrC, KpNifA, RmNifA, andAtVirG have been aligned according to Drummond et al. (7) andWinans et al. (43). The other proteins have been aligned according totheir sequences published in references 4 and 14 (EcTyrR) and 31(RlDctD) and in this paper (PspHrpS). Solid boxes indicate knownDNA binding motifs; open boxes indicate proposed motifs predictedfrom amino acid sequence homologies.

translation experiments support the conclusion drawn fromthe DNA sequence and coding probability analysis thatHrpS lacks significant N-terminal extensions. Furthermore,the regions directly upstream (540 base pairs), as well asdirectly downstream (2 kb), from hrpS were translated in allthree frames, and an additional search of the NBRF-PIRdatabase was carried out. However, no amino acid sequencesimilarities to N- and C-terminal domains of the proteinsmentioned above or to the N-terminal domains of otherregulator proteins, which resemble those of NtrC and DctD(28, 31), were found.A potentially important feature of HrpS is the presence of

a putative ATP-binding site (Fig. 5), also proposed forseveral of the homologous proteins above (31, 42). Further-more, the portion of HrpS between residues 279 and 300showed 40% similarity to the helix-turn-helix motifs (29) ofNtrC proteins located in domain E (7, 31). The possiblesignificance of the similarity of this motif of HrpS to thehelix-turn-helix motif of NtrC and NifA remains to bedetermined.

Regulation of hrpD expression by hrpS. Lindgren et al.(19a) recently characterized another segment of the hrpcluster of P. syringae pv. phaseolicola whose expressionwas shown to be induced during early stages of infection andto remain repressed during in vitro growth. This region isgenetically unlinked to hrpS (19a) and is here designatedhrpD. Expression of the hrpD locus was measured inNPS3121-12 and NPS4006-12 by means of a new reportergene system which involves a promoterless ice nucleationgene (inaZ) inserted into the hrpD gene in the properorientation. Gene expression was quantitatively assayed bymeasuring the increase in ice nucleation activity of thebacteria in planta. The ice nucleation activity of NPS4006-12was considerably lower (10'- ice nuclei per cell) comparedwith NPS3121-12 (10-1 ice nuclei per cell). Therefore, hrpSgene function is required for activation ofhrpD in planta. Wesubsequently transferred the plasmids used in our comple-mentation analysis (Fig. 1) into the NPS4006-12 mutant. Icenucleation activity in planta was considerably greater in thepresence of plasmids pCG4202 and pCG162, which comple-mented strain NPS4006 to Hrp+, but did not change appre-ciably in the presence of pCG4201 and pCG161, which didnot complement the mutant (Fig. 7).The small but reproducible difference in hrpD induction

obtained with pCG4202+ and pCG162+ merodiploids is mostlikely due to different strengths of the promoters which drivehrpS transcription in the two plasmids. In pCG4202, hrpS

VOL. 171, 1989

I

I

A P11-11

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

5036 GRIMM AND PANOPOULOS

Spi ce 1 2

hrpD

4006

Bg H E K

hrpS

FpCG 4202FH

pCG 4201

Bg 0pCG 162 P

pCG 161

H

Bg a+ i

hrpD induction

n/c x 10

410

510

FIG. 7. Induction of hrpD in planta at 24 h postinoculation, assayed by ice nucleation activity of the hrpD12::inaZ fusion in relation to afunctional or mutated hrpS locus. Values are given in nuclei per cell (n/c), rounded to the nearest power of 10. The top line shows a physicalmap of parts of the hrp cluster of P. syringae pv. phaseolicola. Two-transcriptional orientation of hrpS and hrpD genes is indicated belowthe restriction map by thin arrows, according to their transcriptional orientation. The two chromosomal mutations of NPS4006-12 are shownat the top. The Tn3-Spice-12 insertion creating the hrpD::inaZ fusion is oriented from left to right. The Tn5 mutation and the plasmids usedare given as described in Fig. 1.

expression is controlled by a native promoter(s), as dis-cussed earlier. In pCG162, hrpS expression is regulated bythe trp promoter of plasmid pTE3. To compare the relativestrengths of these promoters, we cloned the promoterlessinaZ gene into the EcoRI site E2 of the HindlIl fragmentH1-H2 in right-to-left orientation (pCG45.21) and into plas-mid pTE3 such that inaZ is expressed from the vector trppromoter (pCG8). As shown in Table 1, inaZ expression was1,000-fold higher in P. syringae pv. phaseolicola carryingpCG8 compared with pCG45.21, suggesting that the trppromoter is considerably stronger. The differences in inaZreporter activities between the data in Fig. 7 and Table 1cannot be compared quantitatively for various reasons (19a).However, the higher level of hrpD: :inaZ activation observedin the presence of pCG162 suggests that overexpression ofthe regulatory HrpS protein in plasmid pCG162 led toincreased induction of hrpD.

DISCUSSION

We have determined the nucleotide sequence of a geneticlocus, designated hrpS, of the hrp cluster of P. syringae pv.phaseolicola NPS3121. The locus contains a long ORF(ORF302) capable of coding for a protein of 302 amino acids.Computer analysis based on the Positional Base Preferenceprogram (38) showed a high coding probability for ORF302.Putative regulatory sequences upstream of ORF302 includemotifs that resemble the -24/-12 regions of the promoterrecognized by the minor form of E. coli polymerase (aS4),but no -35/- 10-type promoter sequences, and a hexanucle-otide that resembles closely the Shine-Dalgarno sequencespresent in several E. coli and bacteriophage genes (12). Our

TABLE 1. inaZ expression under different promoter control

Plasmid Promoter INA induction"Plasmid Promoter ~~~~~~(n/cx 10-')

pLAFR6: :inaZ io-3bpCG45.21 N 102pCG8 trp 10s

Measurements of ice nucleation activity (INA) were made in P. svringaepv. phaseolicola NPS3121 during exponential growth in King's B medium asdescribed elsewhere (19a). n/c, Ice nuclei per cell; N, native promoter(s).

b Values have been rounded to the nearest power of 10.

complementation and sequence data indicate that ORF302may be part of a transcriptional unit extending furtherupstream.Our experiments also show that hrpS functions as a

regulatory gene, since it promotes the expression of anotherhrp locus, hrpD, which was previously shown to be acti-vated at early stages of bacterial infection. A regulatory roleis consistent with the extensive similarity between the pre-dicted amino acid sequence of the protein product of hrpS,referred to as the HrpS protein, and the products of severalregulatory genes of enteric and plant symbiotic bacteria.These proteins constitute the so-called NtrC family andrequire the alternate sigma factor (r54) encoded by rpoN ascoactivator (13). RpoN (NtrA) enables the RNA polymeraseholoenzyme to recognize promoters having the consensussequence -26 CTGGYAYR-N4-TTGCA -10 (-24/- 12 pro-moters) rather than the canonical -35/-10 promoters. TheNtrC protein is the most extensively studied member of thisgroup. In the enteric bacteria (E. coli, S. typhimurium, K.pneumoniae), NtrC operates as part of a global controlsystem (the ntr system) which mediates the transcriptionalactivation or repression of many different genes and operons(24, 32, 41). A similar system operates in a variety of species,including Rhizobiulm spp., Bradyrhizobium japonicum,Pseudomonas spp. (41), and Agrobacterium tumefaciens(34). Genes subject to control by the ntr system are involvedin a wide variety of catabolic, biosynthetic, transport, andbehavioral functions (see reference 41 for references). Wesuggest that the HrpS protein is another member of the NtrCfamily of proteins and that genes involved in plant pathogen-esis by P. syringae pv. phaseolicola and other relatedphytopathogens represent another group of genes subject toglobal control by an ntr-like regulatory system.On the basis of segmental homology with other regulatory

proteins, the NifA proteins of R. meliloti and K. pneumoniaehave been divided into four distinct domains, designated A,C, D, and E (7). Similarly, the NtrC protein has been dividedinto four domains, designated B, C, D, and E. Theseproteins share similar sequences, primarily in their C, D, andE domains. Domain D and, possibly, part of domain E alsoshow sequence similarity to HrpS, provisionally making thisprotein the shortest member of the NtrC family of proteins.Although the predicted N terminus of HrpS has not beendirectly confirmed, our sequence and in vitro translation

J. BACTERIOL.

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

LOCUS HOMOLOGY OF P. SYRINGAE AND OTHER PROCARYOTES 5037

data, as well as the nucleotide sequence of a hrpS homologfrom the related pathogen P. syringae pv. savastanoi(Grimm and Panopoulos, manuscript in preparation), indi-cate that the HrpS coding region does not extend beyond theHindIII site Hi. Furthermore, no similarities between thepredicted amino acid sequences from regions directly up-stream and downstream of hrpS and the N or C terminus ofNtrC-related proteins were detected when the current ver-sion of the NBRF-PIR database was specifically searched.

Specific functions have been demonstrated or suggestedfor each of the domains in the NtrC protein family. Forexample, the D domain probably participates in interactionwith the RNA polymerase holoenzyme or the sigma factorRpoN (u54) (7) and is likely to play an important role inactivator function. The stretch of negatively charged resi-dues near the N terminus of HrpS may facilitate interactionswith RpoN or the RNA polymerase holoenzyme, as pro-posed for two activator proteins of yeast (23). Activationmay involve ATP, since the homology between the Ddomain of the NtrC protein group and HrpS includes apotential ATP-binding site in the N-terminal part of HrpS.The N-terminal domain of NtrC serves as a substrate for

phosphorylation by another protein, NtrB, by which theNtrC protein can be switched from repressor to activatorform (17, 27). It was suggested that the N-terminal domainsof NtrC and DctD interact with other proteins, NtrB andDctB, respectively (7, 31). The NtrB-dependent phosphory-lation of NtrC was recently established (17). If proteinsanalogous to NtrB, to DctB, and to the sensor proteins ofother two-component regulatory systems exist in P. syringaepv. phaseolicola and participate in interactions with HrpS,the mechanism involved will likely differ in important re-spects from those of the other members of the NtrC proteinfamily because of the absence of an N-terminal domain inHrpS. Significantly, the N-terminal domains of the NifA inR. meliloti and B. japonicum are dispensable for activatorfunction (2, 9; E. Huala and F. M. Ausubel, J. Bacteriol., inpress).Domain E of NtrC and NifA contains a helix-turn-helix

motif (29) and is involved in DNA binding at or upstream ofRpoN-dependent promoter regions (1, 7, 15, 30). We foundgood similarity between the C terminus of HrpS and theproposed helix-turn-helix motif (E domain) of NtrC. Thismight indicate that HrpS also has sequence-specific DNA-binding functions, although the DNA-binding capacity ofHrpS remains to be determined.Ronson et al. (31-34) speculated that a progenitor gene

encoding the conserved central domain (D) has been adaptedduring evolution for use in several regulatory systems thatrequire RpoN as a coactivator. The amino acid sequencesimilarity revealed in our study leaves little doubt that hrpSis a direct or indirect descendant of the same progenitorgene. Addition of different N-terminal domains (A in NifA, Bin NtrC and DctD, and the unrelated N-terminal domain inTyrR) presumably reflects functional diversification or reg-ulatory optimization. Beynon et al. (2) reported a ca. 100-fold increase in the activator function of the R. meliloti NifAprotein when the A domain was deleted precisely. Thus, theaddition of N-terminal domains during the evolution of theseproteins may also be viewed as a tempering process toreduce, in addition to regulating, the activity of the hypo-thetical progenitor domain D. Since the HrpS protein appar-ently consists only of the central domain D (Fig. 6), theregulatory system that controls genes involved in the patho-genic process and the elicitation of plant resistance re-sponses would appear primitive. Further studies into the

mechanism by which HrpS regulates the expression of hrpDas well as other hrp loci (M. N. Mindrinos, L. G. Rahme,and N. J. Panopoulos, manuscript in preparation) and otherinducible genes involved in the Pseudomonas-plant interac-tions (T. V. Huynh, D. Dahbeck, and B. J. Staskawicz,manuscript submitted) may shed new light on the biochem-ical mechanisms that regulate bacterial gene expressionduring plant pathogenesis.

ACKNOWLEDGMENTS

We thank G. Franz, M. Kokkiudis, and C. Savakis for theirassistance in computer analysis. F. M. Ausubel brought to ourattention the homology of NtrC and NifA to TyrR and unpublisheddata on NifA domain deletions, and S. Henikoff made available to ushis manuscript before publication. We also thank R. Frederick forconstructing the Pss-Lib2 library and for other technical assistanceand inspiring discussions, L. G. Rahme for making available themutants NSP3121-12 and NPS4006-12 for use in this work, and B. J.Staskawicz for making available pLAFR6 and pLAFR6::inaZ. S.Kustu, G. Thireos, F. Kafutos, and F. M. Ausubel provided helpfulcomments during the preparation of the manuscript.

This work was supported in part by grants PCM-8313052 andPCM-8409723 from the National Science Foundation, by postdoc-toral fellowships to C. Grimm from the European Economic Com-munity and the Deutsche Forschungsgemeinschaft, and by theSecretariat of Research and Technology, Greece.

LITERATURE CITED1. Ames, G. F.-L., and K. Nikaido. 1985. Nitrogen regulation in

Salmonella typhimurium: identification of an ntrC protein bind-ing site and definition of a consensus binding sequence. EMBOJ. 4:539-547.

2. Beynon, J. L., M. K. Williams, and F. D. Cannon. 1988.Expression and functional analysis of the Rhizobium melilotinifA gene. EMBO J. 7:7-14.

3. Buikema, W. J., W. W. Szeto, P. V. Lemley, W. H. Orme-Johnson, and F. M. Ausubel. 1985. Nitrogen fixation specificregulatory genes of Klebsiella pneumoniae and Rhizobium me-liloti share homology with the general nitrogen regulatory genentrC of K. pneumoniae. Nucleic Acids Res. 13:4539-4555.

4. Cornish, E. C., V. P. Argyropoulos, J. Pittard, and B. E.Davidson. 1986. Structure of the Escherichia coli K12 regulatorygene tyrR. J. Biol. Chem. 261:403-410.

5. Dale, R. M., B. A. McClure, and J. P. Houchins. 1985. A rapidsingle-stranded cloning strategy for producing a sequential se-ries of overlapping clones for use in DNA sequencing: applica-tion to sequencing the corn mitochondrial 18S DNA. Plasmid13:31-40.

6. Ditta, G., S. Stanfield, D. Corbin, and D. R. Helinski. 1980.Broad host range DNA cloning system for Gram-negativebacteria: construction of a gene bank of Rhizobium meliloti.Proc. Natl. Acad. Sci. USA 27:7347-7351.

7. Drummond, M., P. Whitty, and J. Wootton. 1986. Sequence anddomain relationships of ntrC and nifA from Klebsiella pneumo-niae: homologies to other regulatory proteins. EMBO J. 5:441-447.

8. Engelhoff, T. T., and S. R. Long. 1985. Rhizobium melilotinodulation genes: identification of nodDABC gene products,purification of nodA protein, and expression of nodA in Rhizo-bium meliloti. J. Bacteriol. 164:591-599.

9. Fischer, H. M., T. Burderer, and H. Hennecke. 1988. Essentialand non-essential domains in the Bradyrhizobium japonicumNifA protein: identification of indispensable cysteine residuespotentially involved in redox reactivity and/or metal binding.Nucleic Acids Res. 16:2207-2224.

10. Flor, H. 1971. Current status of the gene-for-gene concept.Annu. Rev. Phytopathol. 9:275-296.

11. Friedman, A. M., S. R. Long, S. E. Brown, W. J. Buikema, andF. M. Ausubel. 1982. Construction of a broad host range cosmidvector and its use in the genetic analysis of Rhizobium mutants.Gene 18:289-296.

VOL. 171, 1989

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

5038 GRIMM AND PANOPOULOS

12. Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B. S. Singer,and G. Stormo. 1981. Translational initiation in prokaryotes.Annu. Rev. Microbiol. 35:365-403.

13. Gussin, G. N., C. W. Ronson, and F. M. Ausubel. 1986.Regulation of nitrogen fixation genes. Annu. Rev. Genet. 20:567-591.

14. Henikoff, S., and J. C. Wallace. 1988. Detection of proteinsimilarities using nucleotide sequence databases. Nucleic AcidsRes. 16:6191-6204.

15. Hirshman, J., P. K. Wong, K. Sei, J. Keener, and S. Kustu.1985. Products of nitrogen regulatory genes ntrA and ntrC ofenteric bacteria activate ginA transcription in vitro: evidencethat the ntrA product is a sigma factor. Proc. Natl. Acad. Sci.USA 82:7525-7529.

16. Jones, R., and R. Haselcorn. 1989. The DNA sequence of theRhodobacter capsulatus ntrA, ntrB, and ntrC gene analoguesrequired for nitrogen fixation. Mol. Gen. Genet. 215:507-516.

17. Keener, Y., and S. Kustu. 1988. Protein kinase and phosphopro-tein phosphatase activities of nitrogen regulatory proteinsNTRB and NTRC of enteric bacteria: roles of the conservedamino-terminal domain of NTRC. Proc. Natl. Acad. Sci. USA85:4976-4980.

18. Khan, H., M. Buck, and R. Dixon. 1986. Deletion loop muta-genesis of the nifL promoter from Klebsiella pneumoniae: roleof the -26 to -12 region in promoter function. Gene 45:281-288.

19. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

19a.Lindgren, P. B., R. Frederick, A. G. Govindarajan, N. J.Panopoulos, B. J. Staskawicz, and S. E. Lindow. 1989. An icenucleation reporter gene system: identification of induciblepathogenicity genes in Pseudomonas syringae pv. phaseolicola.EMBO J. 8:1291-1301.

20. Lindgren, P. B., N. J. Panopoulos, B. J. Staskawicz, and D.Dahlbeck. 1988. Genes required for pathogenicity and hypersen-sitivity are conserved and interchangeable among pathovars ofPseudomonas syringae. Mol. Gen. Genet. 211:499-506.

21. Lindgren, P. B., R. C. Peet, and N. J. Panopoulos. 1986. Genecluster of Pseudomonas syringae pv. "phaseolicola" controlspathogenicity of bean plants and hypersensitivity on nonhostplants. J. Bacteriol. 168:512-522.

22. Lipman, D. J., and W. R. Pearson. 1985. Rapid and sensitiveprotein similarity searches. Science 227:1435-1441.

23. Ma, J., and M. Ptashne. 1987. Deletion analysis of GAL4defines two transcriptional activating segments. Cell 48:847-853.

24. Magasanik, B. 1982. Genetic control of nitrogen assimilation inbacteria. Annu. Rev. Genet. 16:135-168.

25. Melton, D. A., P. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn,and M. R. Green. 1984. Efficient in vitro synthesis of biologi-cally active RNA and RNA hybridization probes from plasmidscontaining a bacteriophage Sp6 promoter. Nucleic Acids Res.12:7035-7056.

26. Nees, D. W., P. A. Stain, and R. A. Ludwig. 1988. TheAzorhizobium calinodans nifA gene: identification of upstream-activating sequences including a new element, the "anaerobox.-" Nucleic Acids Res. 16:9839-9853.

27. Ninfa, A., and B. Magasanik. 1986. Covalent modification of theglnG product NRI by the gInL product NRI, regulates the

transcription of the gInALG operon in Escherichia coli. Proc.Natl. Acad. Sci. USA 83:5909-5913.

28. Nixon, B. T., C. W. Ronson, and F. M. Ausubel. 1986. Two-component regulatory systems responsive to environmentalstimuli share strongly conserved domains with the nitrogenassimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad.Sci. USA 83:7850-7854.

29. Pabo, C. O., and R. T. Sauer. 1984. Protein-DNA recognition.Annu. Rev. Biochem. 53:293-321.

30. Reitzer, L. J., and B. Magasanik. 1986. Transcription of glnA inE. coli is stimulated by activator bound to sites far from thepromoter. Cell 45:785-792.

31. Ronson, C. W., P. M. Astwood, P. M. Nixon, and F. M. Ausubel.1987. Deduced products of C4-dicarboxylate transport regula-tory genes of Rhizobium leguminosarum are homologous tonitrogen regulatory gene products. Nucleic Acids Res. 15:7921-7934.

32. Ronson, C. W., B. T. Nixon, L. M. Albright, and F. M. Ausubel.1987. Rhizobium meliloti ntrA (rpoN) gene is required fordiverse metabolic functions. J. Bacteriol. 169:2424-2431.

33. Ronson, C. W., B. T. Nixon, and F. M. Ausubel. 1987. Con-served domains in bacterial regulatory proteins that respond toenvironmental stimuli. Cell 49:579-581.

34. Rossbach, S., J. Schell, and F. J. de Brujin. 1987. The ntrC geneof Agrobacterium tumefaciens C58 controls glutamine syn-thetase (GSII) activity, growth on nitrate and chromosomal butnot Ti-encoded arginine catabolism pathways. Mol. Gen. Genet.209:419-426.

35. Ruether, U., and B. Mueller-Hill. 1983. Easy identification ofcDNA clones. EMBO J. 2:1791-1794.

36. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

37. Shine, J., and L. Dalgarno. 1974. The 3'-terminal sequence ofEscherichia coli 16S ribosomal RNA: complementarity to non-sense triplets and ribosome binding sites. Proc. Natl. Acad. Sci.USA 77:7117-7121.

38. Staden, R. 1984. Graphic methods to determine the function ofnucleic acid sequences. Nucleic Acids Res. 12:551-567.

39. Staskawicz, B. J., D. Dahlbeck, and N. Keen. 1984. Clonedavirulence gene of Pseudomonas syringae pv. glycinea deter-mines race-specific incompatibility of Glycine max (L.) Merr.Proc. Natl. Acad. Sci. USA 81:6024-6028.

40. Staskawicz, B., D. Dahlbeck, N. Keen, and C. Napoli. 1987.Molecular characterization of cloned avirulence genes from race0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacte-riol. 169:5789-5794.

41. Thony, B., H.-M. Fischer, D. Anthamatten, T. Bruderer, and H.Hennecke. 1987. The symbiotic nitrogen fixation regulatoryoperon (fixRnifA) of Bradyrhizobium japonicum is expressedaerobically and is subject to a novel, nifA-independent type ofactivation. Nucleic Acids Res. 15:8479-8497.

42. Walker, J. E., M. Saraste, M. J. Runswick, and N. J. Gay. 1982.Distantly related sequences in the ot- and P-subunits of ATPsynthase, myosin, kinases and other ATP-requiring enzymesand a common nucleotide binding fold. EMBO J. 1:945-951.

43. Winans, S. C., P. R. Ebert, S. E. Stachel, M. P. Gordon, andE. W. Nester. 1986. A gene essential for Agrobacterium viru-lence is homologous to a family of positive regulatory loci. Proc.Nati. Acad. Sci. USA 83:8278-8282.

J. BACTERIOL.

on April 7, 2018 by guest

http://jb.asm.org/

Dow

nloaded from