JOURNAL OF BACrERIOLOGY, Aug. 1993, p. 5193-5204 Vol. 175, No. 160021-9193/93/165193-12$02.00/0Copyright © 1993, American Society for Microbiology

The Rhizobium meliloti Rhizopine mos Locus Is a MosaicStructure Facilitating Its Symbiotic Regulation

PETER J. MURPHY,`* STEPHAN P. TRENZ,2 WOJCIECH GRZEMSKI,1 FRANS J. DE BRUIJN,2tAND JEFF SCHELL2

Department of Crop Protection, Waite Institute, University ofAdelaide, Glen Osmond, South Australia 5064,Australia, 1 and Max Planck Institut fir Zuchtungsforschung, 5000 Koin 30, Germany2

Received 29 March 1993/Accepted 10 June 1993

The Rhizobium meliloti LS-30 mos locus, encoding biosynthesis of the rhizopine 3-0-methyl-scyllo-inosamine,is shown to be a mosaic structure. The mos locus consists of four open reading frames (ORFs) (ORF1 andmosABC) arranged in an operon structure. Within this locus, several domains of homology with otherprokaryotic symbiotic genes (nitH, fxA,fixU, and nift) are present, suggesting that this locus may representa hot spot for rearrangement of symbiotic genes. Unusually, these domains are present in the coding as well asnoncoding regions of the mos locus. Proteins corresponding to those encoded by mosABC, but not ORF1, havebeen detected in nodule extracts by using antibodies. As ORF1 shows extensive homology with the 5' region ofthe nifH gene (P. J. Murphy, N. Heycke, S. P. Trenz, P. Ratet, F. J. de Bruin, and J. Schell, Proc. Natl. Acad.Sci. USA 85:9133-9137, 1988) and a frameshift mutation indicates that expression of this ORF is not requiredfor mos activity, we propose that the mos locus has acquired a duplicated copy of nijHI, including the promoterregion, in order to become symbiotically regulated. Surprisingly, since the functions are likely different, MosAhas an amino acid sequence similar to that of the DapA protein ofEscherichia coli. The central domain ofMosBhas extensive homology with a range of diverse proteins involved with carbohydrate metabolism in eitherantibiotic or outer-cell-wall biosynthesis. This region is also common to the regulatory proteins DegT and DnrJ,suggesting a regulatory role for MosB. The structure ofMosC is consistent with its being a membrane transportprotein.

Rhizopines are novel symbiotic compounds synthesized innodules during the rhizobium-legume symbiosis and are usedas proprietary growth substrates by the nodule-inciting bac-teria (39). The first rhizopine found was 3-0-methyl-scyllo-inosamine (3-O-MSI) (37, 63), produced in nodules inducedon alfalfa by Rhizobium meliloti L5-30. Rhizopines are notessential for nitrogen fixation or nodulation, as they are notinduced by all rhizobia. Instead, their role is considered tobe the enhancement of the bacterial partner in symbiosis(39). As such, rhizopines can be considered secondarymetabolites of the plant-rhizobium interaction.The genes involved in the synthesis of the rhizopine

3-O-MSI (mos genes) and those involved in its catabolism(moc genes) have been isolated previously (37). They areclosely linked and are on a large bacterial plasmid whichcarries a number of genes essential for symbiotic nitrogenfixation, including nodulation (nod and hsn) and nitrogenfixation (nifandfix) genes (37). moc and mos genes appear tobe differentially regulated, with the mos genes being ex-pressed in the symbiosome when bacteria have differentiatedinto bacteroids and the moc genes, on the other hand, beingexpressed in free-living bacteria. An important finding sup-porting this concept was that expression of the L5-30 moslocus is directly controlled by the symbiotic nitrogen fixationNifA/NtrA regulatory system (38). In this earlier study,DNA sequence analysis of the 5' region of the mos locusrevealed not only consensus motifs for NifA/NtrA binding

* Corresponding author. Electronic mail address: pmurphy@waite.adelaide.edu.au.

t Present address: Michigan State University-Department of En-ergy Plant Research Laboratory and Department of Microbiology,Michigan State University, East Lansing, MI 48824.

but also extensive homology between the leader region ofthe nifHDK operon and the start and first 20 amino acids ofthe NifH protein. In a more recent study, the 5' region of themos locus encoding the production of a closely relatedrhizopine (scyllo-inosamine) induced by R. meliloti Rm220-3was also shown to be symbiotically regulated and havehomology to nifH (53). This differential mode of gene ex-pression between mos and moc genes ensures a beneficialdivision of labor, since the bacteroids would utilize plant-derived substances and energy sources to synthesize acompound which free-living Moc' partner bacteria coulduse as a growth substrate. The unusual structures of the mosloci studied suggest that either part of the nifjH codingdomain is important in rhizopine synthesis or alternativelythe mos genes have acquired the 5' region of a duplicated nifgene, including both regulatory sequences and part of thecoding region. To further investigate these possibilities, wehave sequenced the entire mos region including 1 kb up-stream of the translation start site, analyzed the proteinproducts in vivo and determined whether mos open readingframe 1 (ORF1) codes for an essential protein for rhizopinesynthesis. Our studies show that mos ORF1 does not pro-duce a protein required for rhizopine synthesis but MosA,-B, and -C proteins do appear to be required. MosA is shownto have a high degree of homology with the DapA (dihydro-dipicolinic acid synthase) protein. MosB contains a domainwith extensive homology with regulatory proteins involvedin controlling carbohydrate metabolism, including antibioticand cell wall biosynthesis.

MATERIALS AND METHODS

Strains, plasmids, and DNA manipulation. R melilotiL5-30 (30) is a Moc' Mos' wild-type strain obtained from J.

5193

5194 MURPHY ET AL.

A.

Kpn I BamHI XhoI PstI Bam HI Nsi EcolI I I I I I

ORF 111 kDa

mos A35.8 kDa

Mbo Mbo I

RI Hind III Mlu I Pst_I _

mosB

55.9 kDa

mosC

43.1 (40.9) kDa

fixA nifH Nfel DapA fixA NifT DegT

1 kb

B.

Rm nifH ATGGCAGCTCTGCGTCAGATCGCGTTCTACGGTAAGGGGGGTATCGGCAAGTCCM A A L R Q I A F Y G K G G I G K S

t-Kmpi.mos ORF1 ATGGCAGCTCCGCGTCAGATCGCGTTCTACGGCAAGGGGGGTACCGGCAAGCCCAAGCGAAAGCCTGAGCCGGTAA

M A A P R Q I A F Y G K G G T G K P K R K P E P V

mos ORFiA ATGGCAGCTCCGCGTCAGATCGCGTTCTAC................ GCAAGCCCAAGCGAAAGCCTGAGCCGGTAA

M A A P R Q I A Y A S P S E S L S R *

FIG. 1. (A) Mosaic structure of the mos locus, showing regions of homology to fix4 and nifH genes (hatched boxes). Homology at theamino acid level is shown for Nfe-1, DapA, Niff, and DegT (stippled boxes). Selected restriction enzyme sites referred to in the text are

indicated. Arrow, direction of transcription. (B) Region of mos ORF1 with homology to the nifH coding region and the frameshift mutationcreated at the KpnI site of the mos-containing clone pPM1062.

Denarie. Rm1021 is a Moc- Mos- R meliloti strain (36).Escherichia coli HB101 (5) was used for plasmid transfor-mations. For DNA sequencing, M13mp18 (68) clones were

transformed into E. coli TB1 (2a). Mating of plasmids intorhizobia was accomplished by triparental matings using thehelper plasmid pRK2013 (37). All media, growth conditions,and antibiotic concentrations were as described previously(37). pUC18 (68) was used as a subcloning vector. pPM1062is the broad-host-range cosmid vector pLAFRl with a10.3-kb insert (EcoRI fragments of 3.4 and 6.9 kb) containingthe mos locus from L5-30 (38). pPM1151 is pPM1062 con-

taining a frameshift mutation in ORF1.A frameshift mutation in mos ORF1 was isolated by

cutting pPM1062 at a unique KpnI site situated at the 5' endof ORF1 (see Fig. 1), treating it with T4 DNA polymerase(Boehringer GmbH, Mannheim, Germany), and religating it(54). The size of the deletion was determined by subcloninga 3.4-kb EcoRI fragment containing the modified KjpnI siteinto pUC18 and sequencing across the point of the deletionby using primer 5'-GTGGAAGTGCGCCTTGT-3', whichhybridizes 74 bp downstream.DNA sequencing and analysis. The DNA sequence of the

mos locus and approximately 1 kb upstream was determinedby subcloning four fragments, EcoRI-KpnI (1.0 kb), KpnI-PstI (1.2 kb), PstI-PstI (2.5 kb), and PstI-PstI (1.2 kb), frompPM1062 (Fig. 1A) into M13mp18 and -19 and preparingnested deletions by using ExoIII nuclease and S1 nuclease as

described previously (21). DNA sequencing in both direc-tions was performed on single-stranded templates by thedideoxy method (55) using a-35S-dATP, Klenow fragment,and the M13 oligonucleotide primer. To reduce compres-sions, sequencing reactions were conducted in the presenceof 7-deaza-dGTP nucleotide mixes. DNA sequencing reac-tions were separated by electrophoresis on 5 and 9% poly-acrylamide-7 M urea wedge gradient gels. The sequencesof the junction fragments and the deletion point in the ORF1frameshift mutant were determined by denatured double-stranded DNA sequencing of fragments cloned into plasmidpUC18.DNA and protein sequences were analyzed by the Uni-

versity of Wisconsin Genetics Computer Group program,version 7.0 (9), and by the personal computer program DNAStrider. Protein data base searches were performed witheither the FASTA or the BLAST (1) program. The proteindata bases searched were Swiss-Protein version 23.0, PIRprotein release 34.0, and Genpeptide release 73.1.

Preparation of antibodies. Fusion proteins between the T7protein 10 and mos ORFs were produced by cloning mosDNA fragments in pGEMEX-1 (Promega, Madison, Wis.)and expressing the plasmids in E. coli BL21(DE3) as de-scribed by Studier et al. (60), in the presence of either thepLysS or the pLysE plasmid (Novagene, Madison, Wis.).Fragments were cloned into pGEMEX-1 as follows: ORF1,KpnI-XhoI; ORF2 (MosA), BamHI-BamHI; ORF3 (MosB),

Eco RI Apa l Pst II I

I m I I1- I----

. I . . I II

J. BACTERIOL.

RHIZOBIUM RHIZOPINE mos LOCUS 5195

NsiI-HindIII; ORF4 (MosC) N-terminal hydrophilic region,MboI-MboI; ORF4 (MosC) hydrophobic region, MluI-ApaI(Fig. 1A).The fusion proteins produced in this manner were purified

by sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) (34), cut from gels, homogenized in phos-phate-buffered saline, and mixed 1:1 with Freund's incom-plete adjuvant before being injected into rabbits (NewZealand White). Antibodies were purified by immunoaffinitychromatography on antigen columns (19). Columns wereprepared by binding fusion proteins, partly purified bycentrifugation (60), to Affi-Gel 15 (Bio-Rad, Richmond,Calif.) at pH 7.5 (17).For Western blotting (immunoblotting), nodule protein

extracts were separated by SDS-PAGE (34) using a Bio-RadMini-Protean 1 electrophoresis apparatus. Gels were blottedonto nitrocellulose membranes (Schleicher and Schuell,Dassel, Germany) by using a Bio-Rad Mini Trans-Blotelectrophoretic transfer cell. Blots were probed with purifiedantibodies (14, 19), and bands corresponding to mos proteinswere visualized by the alkaline phosphatase method (14).

Detection of3-0-MSI. To test for mos activity, clones weretransferred from E. coli toR meliloti RmlO21 by triparentalmating. These strains were inoculated onto alfalfa plants(Medicago satliva var. Hunter River) grown on B and D agarin glass test tubes. Nodules were harvested, crushed in H20,and resuspended to a concentration of 2.5 mg of nodules peritl (wt/wt), and the equivalent of 10 mg of nodules wassubjected to high-voltage paper electrophoresis in formic-acetic acid buffer. mos activity is evident, after AgNO3staining, by the presence of a brown spot comigrating withan authentic sample of 3-0-MSI (37, 38).

Nucleotide accession number. The L5-30 mos locus nucle-otide sequence determined in this study has been depositedin the GenBank data base under accession number L17071.

RESULTS

Structure and sequence of the rhizopine synthesis (mos)genes. The mos locus was originally cloned into the broad-host-range cosmid vector pLAFRl as a 10.3-kb regionconsisting of two EcoRI fragments of 3.4 and 6.9 kb (37) andwas later further delimited to a 6-kb EcoRI-PstI fragment(38) (Fig. 1A).The DNA sequence of the 6-kb EcoRI-PstI fragment was

determined for both strands and found to consist of 5,932 bp(Fig. 2). Using the University of Wisconsin Genetics Com-puter Group programs CODONPREFERENCE andTESTCODE (9), four ORFs, all reading in the same direc-tion, were obtained. These ORFs encode polypeptides of thefollowing predicted molecular masses: 11, 36, 56, and 43 (41)kDa; they are designated ORF1 and mosA, -B, and -C,respectively (Fig. 1A).ORF1 is from nucleic acids (na) 984 to 1292. There is a

large gap of 186 bp between ORF1 and mosA (na 1479 to2480), in front of which is a potential Shine-Dalgarno se-quence (56). mosA codes for a protein with a predicted sizeof 35,764 Da. Similarly, there is a large gap near the start ofmosB (na 2770 to 4293), where there are two in-framemethionine residues starting at bases 2731 and 2770, either ofwhich could be the start of mosB. Because there is apotential Shine-Dalgarno sequence in front of the second butnot the first start site, this is considered to be the most likelyinitiation codon. mosB codes for a protein with a predictedsize of 55,850 Da. Twenty-three and eighty base pairs fromthe termination of mosB, there are two in-frame methionine

residues which could initiate mosC (na 4317 [4374] to 5540).The first of these (coding for protein MosCi) is sufficientlyclose (23 bp) to the termination of mosB to enable ribosomalread-through. In front of the second (coding for proteinMosC2) is a potential Shine-Dalgarno sequence, whichwould allow ribosomal reinitiation to produce the shorterprotein. mosCl and mosC2 code for proteins with thepredicted sizes of 43,120 and 40,904 Da, respectively. Aputative Rho-independent terminator, with a region of inter-rupted dyad symmetry followed by thymidine residues (45),which was detected by the program TERMINATOR, issituated approximately 200 bp from the end of mosC.

In vivo protein studies. Gene fusion proteins between theT7 gene 10 and the ORFs shown in Fig. 1 were constructedin the vector pGEMEX-1, and the corresponding fusionproteins were expressed in E. coli. These gave proteins fromgene 10-ORF1, -mosA, -mosB, -mosCi, and -mosC2 of39,435, 59,118, 74,274, 33,642, and 66,618 Da, respectively.After purification of the chimeric proteins, antibodies wereraised in rabbits and further purified by affinity chromatog-raphy. Proteins were extracted from alfalfa nodules inducedbyR melioti L5-30, separated on acrylamide gels, Westernblotted, and probed with the prepared antibodies.The construct consisting of the T7 gene 10 and ORF1 was

designed to exclude the 5' region of ORF1 which showshomology to the nfI- coding region (38) (Fig. 1). Therefore,antibodies to the resulting fusion protein were not expectedto interact with the nitrogenase protein (NifH) in noduleprotein extracts. Figure 3, lane 3, shows that the antibodywhich was raised against the chimeric protein containing theC-terminal domain of the presumptive ORF1 product did notdetect a protein in nodule extracts, although computeranalysis had suggested ORF1 as the most likely readingframe. The electrophoretic conditions used would haveresulted in the small protein (11 kDa) moving just above thebromophenol blue dye marker. The unlikely possibility thatthe antibody did not recognize the antigen was eliminated,because the antibody bound to the chimeric protein onWestern blots and the antibody concentration used to ana-lyze nodule extracts was 1 ,ug/ml, 10 times higher than thatrequired to detect ORF1 fusion protein.

Antibodies prepared to chimeric MosA and MosB proteinsdetected proteins of approximately 36 and 55 kDa, respec-tively, in nodules (Fig. 3, lanes 4 and 5). The molecularweights are in good agreement with the predicted sizes ofproteins deduced from ORFs 2 and 3 (35,764 and 55,850 Da,respectively), indicating that these ORFs are expressed invivo.mosC contains two in-frame potential start sites. Antibod-

ies to the polypeptide encoded between the first and secondstart sites (19 amino acids [aa]; this region is hydrophobic) aswell as to the protein following the second start site (MosC2)were prepared. The antibody to the MosC2 protein detectstwo proteins with the predicted sizes of approximately 41and 43 kDa (Fig. 3, lane 7). The antibody to the hydrophilicregion between the first and second start sites detected thepresence of only one protein, of approximately 43 kDa (Fig.3, lane 6).

Construction of a mos ORF1 frameshift mutant. As de-scribed previously (38), the first 20 aa encoded by ORF1have very extensive homology with the amino-terminal endof the NifH protein, which is the Fe component (or proteinII) of the nitrogenase complex (18). Covering this region andextending for a further 39 aa, there is 74% identity with thenfe-J gene (57) from R. meliloti GR4, a nodulation compet-itiveness gene (Fig. 1). The remaining amino acids deduced

VOL. 175, 1993

5196 MURPHY ET AL. J. BACTERIOL.

1 GAATTCACGTCGCTTCAGTGACGAGCACGATCATGCGCCTTGGGAAGGCGCCGCACGCGCCTTGCCTTCCGATCGCGCAG 80

81 ACAATCCCCGTGCGAGTTGACATGCGCCGCCCTCTCAAAAAATGCCCGCCATTGCAACTGCGGCATTCATCACCTCCGCG 160

161 TCCTATACGCAAAGAAACCAGTCGAGCGGATGATTGCCGCAGCCATATGACATTGTCCGTCGCCTCTGTCGGCCCCTCGA 240

241 CACAGATTGTTCCTTCAAGCATGCGGCCAATTTCCCGATCTAACTATTTGAAAGAAAGCAATTAGCATTATTTCAGTCAC 320

321 GTCTGCGACCTGGCACGACTTTTQCACGATCATCCCCCTAGGAAGCGGTAAGAAGACACAATATCGCGCAAGACAGTGAG 400

401 GACCATTCCGGTTGCTGCTAACGCTTGCCAGCAACGGAAACGCTGGCACACATGTGATTGGAGCCTGGAGAGACACQAT 480

481CA 560

561 QQQQQTQQQQAQQQQTATQAAQQQQQAQ.,T.OQ&QW 640

641 *Q .,GC.JGA,,G,=xAATGCTCTGCCCTCTCCAAAGTACCCGCTATTGCCACTGCGGCATTCTCCGCTCCATCACTTCAGCGCC 720

721 CTAAGCGAAGAAGCCCGGCGTCGTGCGGATGACTGCCGCGTCCATGCGACATTGTCCTTAGCCTTGTCGGCTTTACGACA 800

801 CAGATTGTTCCTCCAACCGTGCGGCCAATTTCCGATCTAACTCTCTGAAAAACAGTCATTAGCATTATTTTAGTAACTCC 880

881 CTCGGCCTGGCOCaCCTTTTSCACGATCAGCCCTGGGCGCGCATGCTGTTGCGCATTCATGTGTCCGAACAACCGAAATA 960ORN 1 *M A A P R Q I A F Y G K G G T G K P K

961 GTTTAAACAACAAAGGAAGCAAGATGGCAGCTCCGCGTCAGATCGCGTTCTACGGCAAGGGGGGTACCGGCAAGCCCAAG 1040

R K P E P V T A S K E D R C L G S P S K N K A H F H S1041 CGAAAGCCTGAGCCGGTAACCGCATCCAAGGAAGATAGATGCCTCGGCTCCCCGTCAAAAAACAAGGCGCACTTCCACAG 1120

R M N V M A R M R G G H G F R V P S A A S R K A M K H1121 CCGCATGAACGTCATGGCTAGAATGCGCGGCGGCCACGGCTTCCGCGTTCCGTCCGCAGCTTCAAGGAAGGCAATGAAAC 1200

K W K G Q P L P K K A L I L L E G T K M G T G Q P P1201 ACAAATGGAAAGGTCAACCACTGCCGAAGAAAGCGCTGATTCTGCTTGAAGGTACTAAAATGGGTACTGGTCAGCCGCCA 1280

V A S *1281 GTGGCGTCATGATCAGCGACTCCTCCGACCCCCGCTTCGATGCAATGCCGTTTGGCGGTTTCAAATATGACGGCATGGAC 1360

1361 CGCGAGGGCGTCCGGTTTGCCTCTGAGGACATGACGCAGCCGAAGGTGGTGTGCATCAACCGCATGAAAACACAGAGCCA 1440MOSMAM F E G S I T A L V T P F A

1441 GGCCTGCTCAAATCCATCCACAATTCA&.GGCAAAGAATGTTTGAGGGTTCGATTACCGCGCTTGTCACGCCTTTTGCT 1520

D D R I D E V A L H D L V E W Q I E E G S F G L V P C1521 GATGATCGCATCGATGAGGTTGCATTGCACGACCTCGTCGAGTGGCAGATCGAAGAGGGATCCTTCGGTCTCGTTCCATG 1600

G T T G E S P T L S K S E H E Q V V E I T I K T A N G1601 CGGGACAACAGGGGAAAGCCCGACGCTCAGCAAATCCGAGCATGAGCAGGTGGTCGAGATCACGATCAAAACCGCAAACG 1680

R V P V I A GAG S N S T A E A I A F V R H A Q N A1681 GGCGTGTGCCCGTCATCGCGGGCGCCGGATCAAACAGCACGGCGGAAGCGATCGCCTTCGTTCGCCATGCGCAGAACGCC 1760

G A D G V L I V S P Y Y N K P T Q E G I Y Q H F K A I1761 GGCGCCGATGGAGTACTGATCGTTTCACCCTATTACAACAAGCCCACCCAGGAGGGGATCTACCAGCATTTCAAGGCGAT 1840

D A A S T I P I I V Y N I P G R S A I E I H V E T L A1841 CGATGCTGCGTCTACTATTCCGATCATCGTCTACAATATCCCTGGCCGTAGCGCCATTGAAATTCACGTTGAAACGTTGG 1920

R I F E D C P N V K G V K D A T G N L L R P S L E R1921 CCCGCATTTTTGAAGATTGCCCGAATGTGAAGGGCGTCAAGGATGCAACCGGCAACCTGTTGCGCCCGTCCCTCGAGCGC 2000

M A C G E D F N L L T G E D G T A L G Y M A H G G H G2001 ATGGCCTGCGGGGAAGACTTCAATCTCTTGACCGGTGAAGACGGCACGGCGCTTGGCTATATGGCGCATGGCGGACATG 2080

FIG. 2. Sequence of the mos operon including 1 kb of the upstream noncoding sequence. The NifA/NtrA binding sites within both the mospromoter (na 885 to 915) and the upstream region homologous to fixA are boldfaced. The deduced amino acid sequence from mos ORF1showing homology to the niflI coding region is doubly underlined. The likely Shine-Dalgarno sequence for each gene is indicated by singleunderlining in front of the ATG start codon. The two regions showing homology to the fixA coding region (na 478 to 589 and 2416 to 2548)are underlined with a continuous line, and where these ORFs continue but have no homology to thefix4 sequence a dashed underline is used.The likely Rho-independent terminator is indicated (< < < > > >).

from this ORF have no significant homology with other whether a protein product from ORF1 is required for mosproteins in the protein data bases searched. activity. Therefore, a frameshift mutation in this ORF wasThe extensive homology between the protein product of constructed. To prepare this construct, we took advantage

mos ORF1 and NifH was surprising and led us to examine of a unique KpnI site in plasmid pPM1062. This site is

VOL. 175, 1993 RHIZOBIUM RHIZOPINE mos LOCUS 5197

C I S V T A N V A P A L C A D F Q Q A C L N G D F A A2081 CTGCATCTCGGTGACGGCGAATGTCGCACCGGCCCTCTGCGCTGACTTCCAGCAGGCCTGCCTGAACGGCGACTTTGCCG 2160

A L K L Q D R L M P L H R A L F L E T N P A G A K Y2161 CCGCCTTGAAACTGCAGGACCGTCTGATGCCGCTGCACCGGGCGCTTTTTCTTGAAACCAACCCGGCGGGGGCGAAATAC 2240

A L Q R L G R M R G D L R L P L V T I S P S F Q E E I2241 GCGCTCCAGCGTCTTGGCCGAATGCGTGGGGATTTGCGCCTACCACTCGTCACGATCAGTCCTTCCTTTCAAGAAGAGAT 2320

D D A M R H A G D P F M M D N A R F A E R I E M D L I2321 CGATGACGCGATGCGCCACGCGGGGGATCCTTTTATGATGGACAACGCAAGATTTGCTGAACGCATCGAGATGGATCTTA 2400

G A N N Q R R K Q G G T C M G L D S G E A P C T S *

2401 TCGGTGCGAACAAC 2480

2481 2QIC, 2560

2561 .,G 2640

2641 * CGGAL.Th, 2720

MO8s *M K V T I R I N G D V

2721 QiAG, TGAACTGCCACTGGTCTTACAGAACCrCAGATAATGAAAGTCACAATTCGCATAAACGGCGACG 2800

L S A Y I P K K D L E E P I I S V A N E D L W G G S2801 TCTTGTCAGCGTATATCCCTAAGAAGGACCTGGAGGAGCCGATCATTTCGGTGGCGAATGAAGACCTATGGGGCGGCTCG 2880

I L L R N G W R L A L P H L P Q E A R L P V T V E A R2881 ATACTGCTGAGGAACGGCTGGCGGCTGGCCCTGCCCCACCTTCCGCAGGAAGCTCGCCTGCCGGTTACCGTCGAGGCAAG 2960

R M F G R L D R G A H E K P D R M T R I G E I S S S Q2961 AAGAATGTTCGGCCGGCTTGATCGAGGTGCGCATGAAAAACCAGATCGGATGACGAGGATCGGTGAGATCTCAAGCTCCC 3040

D A A A M L A E N Q K M H P W P A L T R T A Y E D V3041 AAGATGCCGCTGCAATGCTAGCCGAGAACCAAAAGATGCATCCATGGCCGGCTCTAACGAGAACCGCTTACGAGGATGTC 3120

A A C I S S G E L S G S G L G I I N A F E R R M E E W3121 GCCGCATGTATCTCTTCCGGAGAATTATCCGGATCGGGACTGGGCATTATCAATGCGTTCGAGCGGCGCATGGAGGAGTG 3200

I G G G Y V V S A S S G T A A L T V A L I A L G I Q P3201 GATCGGCGGCGGTTATGTGGTCTCGGCAAGTAGCGGCACAGCCGCCCTGACAGTGGCGCTCATAGCTCTCGGCATCCAGC 3280

G D V V L L P S Y T W A A T A L A P L L I G A I P R3281 CGGGGGACGTTGTGCTTCTGCCTTCCTATACCTGGGCTGCCACAGCATTGGCACCGCTACTTATTGGAGCGATCCCCCGT 3360

F V D I D P N S Y N I S P T A L A A A I T P D V K A I3361 TTCGTCGACATTGATCCGAATTCCTACAACATCTCTCCGACTGCGTTGGCGGCTGCGATCACACCGGACGTCAAGGCAAT 3440

I V V H M H G I S C D M D E I I C H A R E Q G I A V I3441 CATCGTGGTCCACATGCACGGCATTAGCTGCGACATGGATGAGATTATTTGCCATGCGAGAGAGCAGGGCATTGCGGTCA 3520

E D C A Q A H G A L Y K G Q H V G L L S D I G C F S3521 TCGAGGACTGCGCTCAGGCTCATGGCGCACTCTACAAGGGGCAACATGTTGGGCTTCTGTCCGACATCGGATGCTTCAGC 3600

M Q K S K H L S A G D G G F M V T R D P T L A Q K M R3601 ATGCAAAAGAGCAAGCACCTGTCCGCGGGCGATGGCGGATTCATGGTGACAAGGGATCCCACGCTCGCCCAAAAAATGCG 3680

D I C N F G L P T P K A N Y R F D E V V R D G Y A V F3681 CGACATCTGCAATTTCGGTCTCCCCACCCCAAAGGCAAACTACCGATTCGATGAGGTGGTGCGGGACGGTTATGCCGTTT 3760

R E C E Q I G G M F R L Q P M S A A L V M H Q L E H3761 TCCGCGAGTGTGAGCAGATCGGTGGCATGTTCAGGTTACAGCCGATGTCCGCGGCTCTGGTGATGCACCAGCTAGAGCAT 3840

L R Q R I A W L Q T A M E P L V E E S A K I P F F K I3841 CTGCGCCAGCGGATCGCCTGGCTTCAAACTGCAATGGAACCTCTGGTCGAGGAGTCTGCCAAAATCCCTTTCTTCAAGAT 3920

T R S H V D R T H V W H K I R V G I D Y A A V D Y F G3921 TACGCGATCGCACGTCGACCGGACGCATGTATGGCATAAAATCCGCGTTGGAATTGACTATGCGGCAGTCGATTACTTCG 4000

FIG. 2-Continued.

situated near the end of the region with homology to nifH in bp in one of the plasmids was detected. This deletion of 16ORF1 (Fig. 1B). pPM1062 was digested with KpnI and then bp in the ORF1 region with homology to nifH results in antreated with T4 DNA polymerase and religated. A primer ORFi frameshift with consequent termination downstreamwas prepared 74 bp distal of the IYonI site, and the junction of the point where the nifH homology ends (Fig. 1B). Whensite of the religated plasmid was sequenced. A deletion of 16 this plasmid (pPM1151) was introduced into R meliloti

5198 MURPHY ET AL. J. BACTERIOL.

R S M A E I R R G L R N S L A E R G I S S T L W T A4001 GGAGATCAATGGCGGAAATCAGACGGGGGCTGCGCAACAGCCTTGCCGAGCGCGGCATCTCGAGCACGCTGTGGACGGCG 4080

P I L P L Q T A F R P Y A G V V E W T K S A G H L A I4081 CCTATACTCCCACTCCAGACGGCCTTTAGACCCTATGCCGGCGTCGTCGAGTGGACCAAATCCGCTGGCCATCTTGCGAT 4160

E N S F I V F D E N Y P L I A Q E P A K M A E L V V K4161 CGAAAACTCGTTCATCGTCTTCGATGAGAACTACCCGTTGATCGCGCAGGAACCGGCTAAGATGGCGGAGCTTGTTGTCA 4240

MWOC-1L Q Q A W D E H F T S L A R G K * M T

4241 AGCTACAGCAAGCTTGGGACGAGCACTTTACTTCACTCGCTAGAGGCAAGTGACCTTCCGAAGCCGATCCAAATTGATGA 4320MOSC-2 #

R T S P R H H A P S E T K R R V P M G G V H T N P G4321 CCAGGACAAGCCCCCGTCATCATGCTCCAAGCGAAACCAAGCGGAGAGTCCCGATGGGCGGCGTTCATACTAATCCGGGT 4400

K K L T I T A V A F Q F F I N G L V L A A W A T S I P4401 AAAAAGCTAACGATCACGGCTGTAGCCTTTCAGTTCTTTATCAACGGTTTGGTGCTTGCCGCCTGGGCGACAAGCATCCC 4480

H V K N A Y S F N D A E L G V L L L I M A A G A L L F4481 GCATGTTAAAAACGCGTATAGCTTCAACGACGCCGAACTCGGCGTGCTGCTCCTAATCATGGCGGCGGGGGCGTTGCTAT 4560

M S L A G Y F S H V F G S R R M S I Q S A L L F P S4561 TCATGTCGCTAGCGGGTTATTTTTCGCACGTTTTCGGCAGCCGCCGCATGTCGATTCAATCCGCCCTTTTGTTTCCTTCT 4640

A L V L I F A A P N C M T F L C S I V L F G A A N G A4641 GCGCTAGTTCTGATTTTCGCTGCGCCAAACTGCATGACATTCCTCTGCAGCATCGTCCTTTTCGGCGCCGCGAATGGTGC 4720

M D V L M N H Q A K A L E E N G F P R I M A F L H G C4721 GATGGATGTATTGATGAACCACCAGGCCAAGGCGCTCGAAGAGAACGGATTCCCTCGCATTATGGCGTTCCTGCACGGCT 4800

S S T G I L A G I M T F G V I G D G H Y V A R S V T4801 GTTCCAGCACTGGAATACTTGCGGGTATCATGACCTTCGGGGTCATTGGCGATGGCCATTATGTCGCCCGTTCCGTTACC 4880

L L T G I L I V A R W L F P H L L D D V R S G E H R L4881 TTGCTGACCGGTATTTTGATCGTTGCGCGGTGGCTTTTTCCCCACCTTCTGGATGACGTCAGGTCCGGGGAGCACAGACT 4960

A I G E L R N C K L L M F G I L S F L T M V T D G A I4961 CGCGATAGGAGAATTGCGCAATTGCAAGCTCCTTATGTTCGGAATCTTGTCGTTCCTGACAATGGTGACCGACGGTGCAA 5040

A E W S K L Y L I R V E Q V T D Q V G S L G Y V A F5041 TTGCTGAATGGAGCAAGCTCTATCTCATTCGCGTTGAACAGGTGACAGATCAAGTCGGCTCTTTAGGATATGTAGCCTTC 5120

T L L M I A G R I S G D R V K D A I G C R A L I A I S5121 ACGCTCCTTATGATTGCCGGCCGGATCTCGGGAGATCGGGTCAAAGACGCCATCGGATGTCGGGCACTGATAGCGATCAG 5200

G S L A S A G M T T A L F M P S F A G K L A G F A L L5201 CGGGAGCCTTGCGTCGGCGGGCATGACTACGGCTTTGTTCATGCCAAGCTTTGCCGGGAAGCTTGCTGGTTTCGCCCTCT 5280

G L G M A N L V P I I F S E A A S M N T V S K T V G5281 TGGGACTGGGTATGGCCAATCTGGTGCCGATCATCTTCAGCGAAGCCGCTAGCATGAATACTGTGTCGAAGACGGTCGGG 5360

L T F V S V C G Y S G F L V G P P I I G A S R R P L G5361 CTCACCTTCGTTTCGGTCTGCGGCTACTCCGGGTTTCTGGTTGGTCCGCCCATCATTGGCGCATCGCGGAGGCCGTTGGG 5440

S G E L C S S S F A V G V I V A C A S V F F D R H R S5441 CTCGGGCGAGCTTTGCTCTTCATCATTCGCGGTCGGAGTGATCGTAGCTTGTGCTTCGGTATTCTTCGACCGTCACAGAT 5520

G Q P E P *5521 CAGGCCAGCCAGAGCCTTGATCATCCCATGCGCGAACCCTTGGCGCCCACTTCATGGCCGCTTGCATATCACCACCAATG 5600

5601 GGAGCTTAGCAATGAAACGACGAGAGCTACTGTTCCGCGCTGTGGCAGTTGTCACCGCAGTGGTTGCCAACAGGACCACC 5680

5681 CATGGCGAGGAGGAGGGCCTTCTGCCAAAGACGACCAGACGCGATGGCGTTCGCAAAGTCCTCACACAAGGCCAGGAGGA 5760

5761 GATCCTGAAGGCGGTTTTTGATCGTCTGATTCCTGGAGATGAGTTGGGGCCATCGGCATCGGGAGCGGGCTGTCTTGACT 5840

5841 TCCTCGACGACCAACTGGCGGGAGGATATGGCGAGGGTTCGACGATCTATCGAGATGGGCCCGTGCAGCCCCACGAAGAG 5920

5921 CAGATGCTGCAG 5932

FIG. 2-Continued.

RmlO21 and the resulting strain was inoculated onto plants, Putative functions of MosABC proteins. In order to gainrhizopine production was found. We conclude that a protein insight into the possible functions of the proteins encoded byproduct of ORF1 is not required for rhizopine production. mosA, -B, and -C, the deduced amino acid sequences wereThis is further supported by the observation that antibodies scanned against protein data bases by the FASTA andagainst this region did not detect a protein in vivo. BLAST programs.

RHIZOBIUM RHIZOPINE mos LOCUS 5199

97400 1* ~1 l_ t 00* jt

66200

45000~~~~~~~~~.

12 3 4 5 6 7FIG. 3. Western blot of nodule protein extracts Protein extracts

from nodules induced by R meloti L5 30 were probed withantibodies to mos ORFs. Lane 1, marker proteins (Bio-Radlow.molecular-weight markers) stained with Coomassie blue; lane 2,total proteins from nodules stained with Coomassie blue; lanes 3 to7, total nodule protein probed with antibodies to the C-terminalregion of the deduced protein from ORMi (lane 3), MosA (lane 4),MosB (lane 5), the hydrophihic N-terminal region of MosC (lane 6),and the hydrophobic region of MosC (lane 7).

(i) MosA. The MosA protein was found to have extensivehomology (44.3% identity over the entire length of 291 aa)with the DapA (48) protein from E. coli (Fig. 4). Consider-able homology with the dapA gene product from Corynebac-terium glutamicum (34.9%) (Fig. 4) and wheat (31% over 310aa including a number of gaps [data not shown]) was alsoobserved. dapA codes for the enzyme dihydrodipicolinatesynthetase, which is the first enzyme in the diaminopimelateand lysine biosynthetic pathway. This enzyme condensespyruvate and aspartic semialdehyde to 2,3-dihydrodipi-colinic acid. In addition, a 29.1% identity was observed for a289-aa domain of MosA and the E. coli nanA gene product(data not shown) which cleaves, in a reversible reaction,N-acetylneuraminic acid to pyruvate and N-acetyl-D-man-nosamine (28, 41).

(ii) MosB. Comparison of the MosB protein with theprotein data bases indicated that this protein contains twodomains which have distinct homologies with known pro-teins. The first domain is at the N-terminal end of MosB andhas 35.3% homology over 65 aa with the Niff protein fromKiebsiella pneumoniae (2, 4) and 37.1% identity over 62 aawith Niff fromAzotobacter vinelandii (24) (Fig. 5A). In boththese species, nifT codes for a small protein of 72 aaimmediately distal to, and arising from the same transcriptas, the nifHDK operon. There is no known function for theNiff protein (24, 46). Similarly, extensive homology (58.6%identity over 70 aa) between the same region of MosB andthe fixU gene product from Rhizobium leguminosarum bv.trifolii was found (Fig. 5B). fixU is found in an intergenicregion between nifB and nodT (35). The protein productencoded by fixU is predicted to be 70 aa long with amolecular mass of 8 kDa and has no known function. It isclosely linked to what is thought to be a ferredoxin-like gene(fdxN). FixU also has 62% similarity with a previously

MOSA 1.......... . E[F|EL| V TPLjA D . D RIDE HDHFLV E W Q I EEJS F GDAPAEc 1 . . . . . . . . . . .L FITIS I V A|I|V T P DEnKrINV C R KKJIK DrYHifVA S|G|T S ADAPACg 1 M S T G L T A K T G V E HIG TV G V A FT GD D I A E VA ALVLLD KG L D S

MOSA 38 VP CG T T G Z S P T L1S K S E QIV V1E I I K TrIG RVP V I A G A SNS|T A E A I|A FDAPAEc 39 JVIS V G T T G Z S LTLN H D ZNJHA DV VI M T L D LAJD I P V I A G|T|G|A|N A|T A E AIISTDAPACg 51 ~jJATGSTTA~KELAREGjJKL[ V~T~NRTVEDAPA~~~g 5ILVL A.G T T G Z S P TT T A A ZK L E L L K A V R E E V G DA KLI A G VGTN TJ TS V ES

MOSA 88 V R H[Q N G A D G VIr_ A T I I IP GDAPAEc 89 T Q R F N D GI VGICL TV T P Y Y NFRP S Q Z G L Y O R F K A I AlE H|T|D L PQI L Y NVP[URDAPACg 101 A E A[EA SG A D GL LV_E VI

MOSA 138 AIEMH V|E T L A I F E D C P N V KG D A T G N L LPS L E R M A C G E D F NIL LDAPAEc 139 TIGIC D L L PI TV GIRI.A K VKN IIG A T G N LTRN Q I K E L V S DD FVIL LDAPACg 151 E PR[ESTM . tR S E L LA A A T S L I K E T G LAWY[

MOSA 188 DG T G Y C I S V TIA1N V A A L C D F Q Q A C L NG D F A A L K L Q DLDAPAEc 188 D LDFM L GG HG|V|I S VTIN V AAR D MAQ M C K L AAE V| QDAPACg 198 NP L L V WL I SVIF I G H A A P T A L R E L Y T S F EIE G D L V RA RE N A K

MOSA 238 R FLTIN PJA GA |YL Q R M R G. PL V T S PF Q E I DDDAPAEc 238 P LHNLV PIN -P I P V WIAIC K E L V A T DTLRL M T PT DG RZT V R ADAPACg 248 P LVAQG R L G G V S L . A S G I N V G D R L P I M A P N E Q E LZA L R E

MOSA 287 [AD PDAPAEc 288 HAG.DAPACg 298 A G V L

FIG. 4. Alignment of the deduced amino acid sequences of MosA, DapA from E. coli (DAPAEc), and DapA from Corynebacteriumglutamicum (DAPACg). Sequence homologies were determined by the program FASTA, and alignment was derived with the PILEUPprogram (9). Regions of identity between at least two sequences are boxed; residues that are identical for all three sequences are boldfaced.

VOL. 175, 1993

5200 MURPHY ET AL.

A.

MOSB 1 . . M K IWI N D V L S PIV A N E D W G G S L L R N GNIFTKp 1 . M P I VR E R A D L Y A[ODLEFTR I E H NLDA WGGA ES L E G GjRNIFTAv 1 M P S V1 RON D E G Q L T Y I AX K DIg EI L E H D SPE W G G E V T L G D G S

MOSB 47 A L P H L P Q E A R[ L P V R M F G RNIFTKp 50 Y VHPQPG RV PIILJR A T R N T L INIFTAv 51 F I E I P . QLK LL P IT V R A K R A G E A

697272

B.

MOSB 1 V T I NG VL S ArI P K K DLE I I S A N E D L W G GS I LR G WRFIXU 1 IM K A M I R R SIGV G L SIIB S K K V V A VE N E D L W G G F I L V R N G W L G D

MOSB 51 L P Q E A L P V T V E A R R M F G R L D R 72FIXU 51 L P Q G T R L P I T V E A M K Q P D Q D . . 70FIG. 5. Alignment of proteins to the deduced amino acid sequence of the amino-terminal region of MosB. Sequence homologies were

determined by the program FASTA, and alignment was derived with the PILEUP program (9). (A) MosB, NifT from K pneumoniae(NIFTKp), and Niff from A. vinelandii (NIFTAv). Regions of identity between at least two sequences are boxed; residues that are identicalfor all three sequences are boldfaced. (B) MosB and FixU. Identical residues are boxed.

unidentified ORF located in the nif/fix cluster from R.meliloti (29).The central domain of MosB (approximately aa 138 to 308)

has extensive homology with seven other proteins. There is40.8% identity over a 174-aa region with DegT, 39.5%identity over 177 aa with EryC1, 38.6% identity over 171 aawith 0299 from E. coli, 37.6% identity over 179 aa withDnrJ, 37.1% identity over 170 aa with Prg-1, 36.9% identityover 170 aa with StrS, and 33.2% identity over 184 aa withORF10.4 from Salmonella typhimunum (Fig. 6). The regionrevealing the most significant degree of homology betweenMosB and these proteins is shown in Fig. 6 and was deducedby analysis using the FASTA or BLAST program, and thenall eight sequences were aligned by the program PILEUP.The homology extends further, especially if conservedchanges are taken into consideration. This is most noticeablebetween proteins DegT, EryC1, DnrJ, and StrS (61).

In the central region of MosB (aa 256 to 275), a putativehelix-turn-helix motifwas found. The other proteins showinghomology to the central region of MosB also have this motif(Fig. 7) (61, 62).

(iii) MosC. MosC does not have any significant homologywith any of the proteins in the data bases searched. How-ever, it is a very hydrophobic protein with 12 potentialmembrane-spanning regions (Fig. 8). This is typical of mem-brane proteins involved in transport of sugars (26). Such aprotein could be involved in either transport of a precursorfrom the plant into the bacteroid or transport of the rhizopineout of the bacteroid.mos homology to R. melilotifix4. There are two regions in

the mos sequence which have homology with the fix4 genefrom R meliloti (13). One region is located between nucle-otides 1 and 589 of the mos sequence. This region shows97.6% identity with the fixA sequence, including the ex-tended 5' region, NifA/NtrA promoter motifs, leader se-quence, and the first 112 nucleotides of the fix4 ORF. ThisORF then continues for a further 59 nucleotides, showing nosimilarity to the fix;4 coding region. If expressed, this ORFwould code for a small protein predicted to be 6,170 Da insize (Fig. 2).The second region lies between nucleotides 2416 and 2548

of the mos sequence and shows 78.3% identity withfixA overa 135-bp region. The homologous region includes part of theleader (but not the promoter) and the first 81 nucleotides ofthe fixA coding region. This ORF continues for a further 183nucleotides beyond the region of fixA homology beforereaching a TGA stop codon. If expressed, this ORF wouldcode for a small protein of 9,552 Da. The region is incorpo-rated in the C-terminal end of MosA, with the ORF producedbeing in a reading frame different from that of MosA, andcontinues into the intercistronic region between mosA andmosB, ending 38 nucleotides before the start of mosB.

DISCUSSION

The rhizobial rhizopine synthesis (mos) locus consists offour ORFs arranged in an operon structure. Antibody stud-ies indicate that ORFs 2, 3, and 4 are expressed in nodulesbut ORFi is either not expressed or expressed at very lowlevels. Therefore, we have termed these ORFs ORFi andmosA, -B, and -C, respectively. Previously (38), we reportedthat the mos promoter and the 5' region of ORFi have a highdegree of homology to the nWfH 5' region. Results presentedhere indicate that a frameshift mutation preventing theexpression of ORFi still enables the production of therhizopine. Intriguingly, in the wild-type strain we could notdetect a protein encoded by ORFi in nodules. This proteineither is not expressed, is expressed at very low levels, or israpidly degraded. Together, these data support the notionthat the mos locus has acquired a duplicated copy of asymbiotic promoter enabling these genes to be expressed inthe bacteroid and be coordinately regulated with nitrogenfixation genes.The MosA protein has extensive homology over its entire

length with the dapA gene products (48) from E. coli,Corynebacterium sp., and wheat (identities, 44, 35, and 31%,respectively). This enzyme, dihydrodipicolinate synthetase,condenses pyruvate and aspartic semialdehyde to 2,3-dihy-drodipicolinic acid and is the first step in lysine biosynthesis.Interestingly, MosA also has 29% identity over its entirelength with NanA (28, 41) from E. coli, which cleaves,reversibly, N-acetylneuraminic acid to pyruvate and

J. BACTERIOL.

RHIZOBIUM RHIZOPINE mos LOCUS 5201

138 ERR M E E W ISG G Y V[s ASC G T A ALT V A L I42 E A D I A A Y S R AKHDGI G N D AL H IA L Q A42 E A E F A A Y C E N AMH E2T V G G C D AL E LS L V AL.42 E E E F A A Y H G L P Y C T G V DGTN A LV L G L R A L .

E A A L -A A G Q G V EE|A A V S TG T A AV H L A L H A L .

16 D H .. ..]G V R Y AW T F N S G T S A L LA A Y F A L .

37 Q Q W L E Q R F G S A K V L L T P ST ASLE M A A L L .

68 [K K L G E F I G V P@V L T T T S G S SA N LL A T A L T

. |- -L.~~~~GII~PFG;DLVVL LP S.... . . . . .... G V G P G D E V I T T A

... .. .. G V G~F2]G D E I oV P S1.G...GMIGPGDEVV Ty S. . . . . DV G P G D E V T

.GVR.E.. . . G(E AAG P A.D.I..QP G D E

S P K L G E R A L K P G D E V I T V A

MOSB 180 Y TA A1 L A P L L I G A II|D I N S Y D I MDEGT 94 F T F F A T A G S I A R A G A K P V F V D I D Pl VT F N I D P A Q VIE A ArV7TE K T X A I I P V H LERYC1 94 H T F I A T L G V P V G A V P VVEP E G V S H TLD P A L VEQIJP R TAAI L P V R LDNRJ 94 N TA A V V A I D A V G A T P V F V DVH E E N Y L M DT G R L R S V I GP R TR C L L P V H LSTRS - H T FI GS AS P V T Y LIG A R P V FID V DF]H C L D D S V K S L I R T K A IVV V HI

PRG1 52 L T YHFAAL S P V F A L R G D VVLVD D P V S R G LD PK A L E A A I N T R V V T VV H Q0299 79 Y T FV S TAN A F V L R G AK IV F V DVPDTM N IDET L I EAAI T KIT IVV Y

ORF10.4 118 A P T TVN P A I Q N IPV F V D V P T Y N I D A S L I EA S K A I MAH T

MOSB 230 H G I S C EMI C H A R E Q I A V I D C AQAH[A'LKQ HV GL LSDIGCF MDEGT 144 Y G Q M A D AAjAA I|A|K RrWVGV I Z DMj A Q A|I|G A KNIGAK C|V G|E L GTA A TYSERYC1 143 Y G H P A D L RAJ IIAID RIH G L A LVI DLVJAQ AQVAIVGARH RAIRH RV GHRG S N A A A F . IF

DNRJ 144 Y G Q S V DMTV L E L|AIA E H K Z D C A Q A|H|G A R R H|G|R LVG Q AAF| FISTRS - N G I A A D M ALTEVAA E GVP V I DrA A GTE I G G R PIGG FG D A C

VS LWPRG1 102 W G H P CLD MDAGLG VVAGER Y LGR VLZD CS H AIH SSR YK||K PVG TFG D A A VFS . L

0299 129 A G V A C EMD MELAK KNL IVFVVZ DrA A G V M S T|Y|K|G|R ALG T I GHI G CF SORF10.4 168 L

GN A F N L S E V R R ILA D K Y NLj W L I Z D C C

DA ELQT T F I GT* F Y

MOSB 280 Q K SrHYSr .IG D G GF MTRRP TK MMRD ICDEGT 194 F P TIKINILIGIAIYGGD G GIM I IIT NIDID EL A K C RVIRERYC1 193 Y P GIKIN LIG DAGDA V V TVTIDIPAL A E RII RL RDNRJ 194 Y P TIKIVLWJG AJYG D G G A V V T P QA EV D RL RR L R

STRS - F E QIKIV I T S G GEG G A VLTD N P V Y ERIVRRLRPRG1 151 Q A N|K|A V YA .GIEIG G LV DDA L V Q RA T LL0299 178 H E T KN Y TALJGG E G G ATL I NDJK ALIE RA E I IRORF10.4 218 P A H H I . . T MME|G A VF []K S G E K K I. I E S FS

FIG. 6. Alignment of proteins DegT, EryC1, DnrJ, StrS, Prg-1, 0299 (E. coli), and ORF10.4 (S. typhimurium) to the deduced amino acidsequence of the central region of MosB. Sequence homologies were determined by the program BLAST (1), and alignment was derived withthe PILEUP program (9). The sequence for StrS was taken from the report of Stutzman-Engwall et al. (61). Regions of identity between atleast four sequences are boxed; residues that are identical for all eight sequences are boldfaced. A putative helix-turn-helix region is indicated(asterisks).

N-acetyl-D-mannosamine. Both these enzymes utilize pyru-vate as a substrate, and initially we thought (39) that MosAmay be involved in the condensation of pyruvate to inositolderivatives by an unknown pathway. However, more re-cently we described the mos locus from another strain ofRmeliloti, strain Rm220-3. This bacterium induces the produc-tion in nodules of a rhizopine, scyllo-inosamine, which ischemically very similar to 3-O-MSI and is regulated in amanner identical to that of the L5-30 mos locus (53). Hybrid-ization studies with probes from the [5-30 mos locus, as well

* *

1 23 45 67 8

as restriction studies, indicate that mosB and mosC arepresent but mosA is absent from this strain. scyllo-Inosamine differs from 3-0-MSI only by the absence of amethyl group; therefore, we conclude that the likely functionof mosA is to add a methyl group to scyllo-inosamine in theproduction of 3-0-MSI. In light of this finding, it is surprisingthat there is such extensive homology with the condensationenzyme DapA. Although uncommon, distinct enzymaticfunctions for proteins which have similar structures at theamino acid level have been reported, suggesting the likely

* *

9 10 11 12 13 14 15 16 17 18 19 20

Q A H G A L YQ A I G A K YQ A V G A R HQ A H G Al R RQ A L G T E ID A L G T T YH A H G S R YQ G V N S T Y

KNRHGEKK

Q T K T A K D LQ A E A Q K VR A E IA Q R L

GGGGGGG

G

GG

Q H V G L[LSK C V G EJ GH R V G A G SR L V G T Q GR P I G G F GQ M V G T F GK P V G T F GR A L G T H G

E Y Q S A I NT T Q Q S I EIfl R S P N A A

FIG. 7. Possible helix-turn-helix domain in MosB aligned with similar regions within proteins showing extensive homology to the centraldomain of MosB. Both DegT and DnrJ have been assigned regulatory roles on the basis of genetic studies. XCro, 434Rep, and LexA are

known DNA-binding proteins with helix-turn-helix motifs. Important regions where there are small or generally hydrophobic residues (43) are

indicated (asterisks). Residues which comply with these conditions are boxed.

MOSBDEGTERYClDNRJSTRSPRG10299ORF10.4

MOSBDEGTERYC1DNRJSTRSORF10 .4PRG10299

)Cro434REPLEXA

D I GCT A ATN A AAH A AAD L ACD I GTD A AVH I G C

K A I HQ L ENE E H L

VOL. 175, 1993

5202 MURPHY ET AL.

100 300

3 v--

2-

-2-3 4 5 6 7 8 9 1011

100 200 300 400

FIG. 8. Hydrophobicity profile of MosC derived with the pro-gram DNA Strider by the procedure of Kyte and Doolittle (32). Thedata are averages for 11 amino acids. The 12 hydrophobic regionswhich are likely membrane-spanning regions are numbered.

importance of domains within proteins. For example, man-delate racemase and the muconate lactonizing enzyme fromPseudomonas putida have similar primary structures (iden-tity, 26%) and very similar secondary, tertiary, and quater-nary structures as determined by X-ray crystallography butcatabolize distinct chemical reactions (40).The central domain of MosB has a region of homology

covering 170 aa with seven other proteins (with identities inparentheses): DegT (41%), EryCl (40%), 0299 from E. coli(38%), DnrJ (38%), Prg-1 (37%), StrS (37%), and theORF10.4 protein from S. typhimurium (33%). Six of theseproteins have roles in carbohydrate metabolism in eitherantibiotic or outer-cell-wall biosynthesis; the seventh, DegT,regulates a diverse range of functions. Of these, DegT andDnrJ have been shown to be regulatory proteins by geneticstudies. DegT from Bacillus stearothermophilus is a single-component pleiotropic regulatory protein (but with no ho-mology to LysR regulatory proteins [22]) effecting alkalinephosphatase activity, flagellum development, and sporula-tion patterns and can substitute for the two-componentregulatory proteins DegS and DegU in Bacillus subtilis (62).It has been proposed that this protein both senses anenvironmental stimulus and activates transcription of genesin response to this. On the other hand, DnrJ and its linkedcomponent DnrI (from Streptomyces peucetius [61]) appearto be members of the two-component regulatory system (51,58, 59) and were characterized by their abilities to overpro-duce intermediates in the antibiotic daunorubicin biosyn-thetic pathway in Streptomyces sp. Prg-1 (from Streptomy-ces alboniger [33]) and StrS (from Streptomycesgriseus [11])are encoded by genes located in clusters for the biosyntheticpathways of the antibiotics puromycin and streptomycin,respectively. EryCl is involved in the biosynthesis of theantibiotic erythromycin in Saccharopolyspora erythraea(previously called Streptomyces erythreus [10]). The genesencoding 0299 from E. coli (7) and ORF10.4 from S.typhimurium (25) are located within clusters involved in thebiosynthesis of the 0 antigen and common antigens, respec-tively, as part of outer-cell-wall polysaccharide biosynthesis.In all these cases, the similarity at the amino acid level withthe regulatory proteins DegT and DnrJ suggests that theymay have regulatory roles (33, 61).These proteins have only limited homology to the two-

component regulatory proteins (for example, some of thefeatures characteristic of the protein kinase-sensor compo-nents are present even though none of the sequences show asignificant overall or regional similarity to such kinases [33,61] [Fig. 6]), and there is no similarity with LysR-typeactivators. However, a role in regulation is strengthened bythe presence of a putative helix-turn-helix structure (Fig. 7).The high degree of variation commonly found for such sites(6, 20, 42, 43) is observed, but the most highly conserved

residues, including a glycine in the turn (position 9) andseveral hydrophobic residues (Fig. 7), are present. Whetherthese proteins have unique features common among proteinsinvolved in carbohydrate regulation is yet to be determined.A further consideration with MosB is that, as a substantialportion of the protein lies outside this regulatory domain,there is a possibility that it may also have an enzymaticfunction.A regulatory role for MosB could be to control housekeep-

ing genes within the nodule which are involved in thebiosynthesis of the rhizopine backbone. A candidate for thispathway could be similar to that in Streptomyces gleboseus,in which myo-inositol is synthesized to streptidine as part ofthe biosynthesis of streptomycin (65). In this context, it isinteresting that there is extensive homology between MosBand the streptomycin regulatory protein StrS.MosC has two potential start sites, and our results using

antibodies suggest that both of these function in vivo, onepresumably from read-through from mosB and the otherfrom reinitiation at a Shine-Dalgarno sequence. Alterna-tively, the protein may be processed, but this is considereda less likely explanation, as there is no obvious signature inthe sequence characteristic of signal peptides (64). MosC hasno significant homology to proteins in the protein data bases.Its structure is that of a very hydrophobic protein with 12putative membrane-spanning regions. This is characteristicof proteins involved in sugar transport across membranessuch as the lactose permease from E. coil, which is involvedin translocation of 3-galactosides (16, 26). MosC could eithertransport a precursor for rhizopine biosynthesis into bac-teroids, where the rhizopine is synthesized, or alternatively,export the finished product from the bacteroid. Rhizopinesecretion and its distribution and localization within thenodule are being investigated in our laboratory to helpanswer this question.

Interestingly, mos ORFi has a region of homology withthe nfe-1 gene from R. meliloti GR4 (57). The nfe regulon isinvolved in nodulation competition (although the basis ofthis is unknown) and is controlled by NifA/NtrA, as is themos locus. Since mos ORFi does not appear to be expressedin L5-30, the importance of this region is unclear.The mos region is interspersed with zones showing homol-

ogy to fragments of symbiotic nitrogen fixation genes (fixA,nifH, niftT, and fixU [Fig. 1A]). Some of these gene frag-ments have promoter elements and are present both withinthe mos coding regions and in the intercistronic regions.Portions of duplicated symbiotic genes, with and withoutpromoters, have previously been found in the vicinity offunctional symbiotic genes in R. meliloti (3, 13) and in R.leguminosarum bv. trifolii (66), although it is unusual to findthem within coding regions of another locus. Where thereare duplicated promoters and amino-terminal regions ofsymbiotic genes, the ORFs produced usually continue for ashort distance after diverging from the symbiotic sequence.Although in a number of cases transcripts are produced fromthese, there is no evidence of translation of these regions (3).The mos region seems to be a hot spot for rearrangements.

How these occur is not clearly understood. It is known thatrearrangements giving rise to duplications and deletions inthe Sym plasmid are common in rhizobia. Some of theseresult in the duplication of very large fragments (50). Indi-vidual genes are also duplicated: for example, several copiesof nodD (23, 49) and nifH (47) per genome exist. Thepresence of reiterated DNA sequences has been implicatedin genetic rearrangements in many microorganisms (44)including Rhizobium (15) and Bradyrhizobium (27) spp. Such

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RHIZOBIUM RHIZOPINE mos LOCUS 5203

repeated sequences could be genes previously duplicated,other reiterated DNA such as rhizobial insertion elementsISRml (52), ISRm2 (12), and ISRm3 (67), or multiple copiesor reiterated sequences without recognizable structural re-gions of insertion elements such as has been found forRhizobium fredii (31) and other rhizobial isolates (8).The mos locus has a distinct mosaic structure both in

terms of homologies with known proteins, those involved insymbiotic nitrogen fixation as well as others, and in terms ofthe functions these proteins encode. Our model consists of alocus which has acquired a symbiotically regulated pro-moter, thus enabling the gene products to be producedduring symbiosis. The MosA, -B. and -C proteins have likelyroles in enzymatic, regulatory, and transport functions,respectively, resulting in the biosynthesis of the rhizopine.This structural organization would represent an efficient wayto utilize pathways and processes already present in thenodule.

ACKNOWLEDGMENTS

We thank N. Heycke for technical assistance; J. Groom, A.Dunbar, and N. Schussler for art and photographic work; and M.Wexler for helpful suggestions with the manuscript.

This work was supported by grants from the Australian WoolCorporation (UAD16), Australian Research Council, and the MaxPlanck Institut. W.G. was supported by an RIRDC fellowship.

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