deduced products of c4-dicarboxylate transport regulatory genes of

Volume 15 Number 19 1987 Nucleic Acids Research

Deduced products of C4-dtearboxylale transport regulatory genes of Rhizobium Uguminosarumare homologous to nitrogen regulatory gene products

Clive W.Ronson1-", Patricia M.Astwood1, B.Tracy Nixon1-4 and Frederick M.Ausubcl2

•Grasslands Division, Department of Scientific and Industrial Research, Private Bag, PalmerstonNorth, New Zealand and 'Department of Genetics, Harvard Medical School and Department ofMolecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA

Received August 10, 1987; Accepted September 9, 1987

ABSTRACTWe have sequenced two genes dctB and dctD required for the activation of theC.-dicarboxylate transport structural gene dctA in free-living Rhizobiumlequmlnosarum. The hydropathic profile of the dctB gene product (DctB)suggested that its N-tenninal region may be located in the periplasm and itsC-terminal region in the cytoplasm. The C-terminal region of DctB wasstrongly conserved with similar regions of the products of several regulatorygenes that may act a6 environmental sensors, including ntrB, envZ, virA, phoR,cpxA, and phoM. The N-terminal domain of the dctD gene product (DctD) wasstrongly conserved with N-terminal domains of the products of severalregulatory genes thought to be transcriptional activators, including ntrC,ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions ofDctD were strongly conserved with the products of ntrC and nlfA,transcriptional activators that require the alternate sigma factor rpoN (ntrA)as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that showthat rpoN product is required for dctA activation, and suggest that DctB plusDctD-mediated transcriptional activation of dctA may be mechanisticallysimilar to NtrB plus NtrC-mediated activation of glnA in E. coli.

PrmuOUlTi.CN

The C4-dicarboxylate transport structural gene dctA is expressed in both

free-living and symbiotic forms of Rhizobium leguminosarum. In free-living

cells, expression of dctA is induced by succinate, fumarate or malate, and

dctA product (DctA) is required for growth on these C4-dicarboxylates as

carbon sources. DctA is also essential for symbiotic nitrogen fixation, as

dctA mutants form nodules that contain bacteroids but do not fix nitrogen

(1-4). The symbiotic role of DctA has not been defined, but it is likely that

DctA is required for transport of C4-dicarboxylate6 into bacteroids for use as

energy substrates (reviewed in 5,6).

The dctA gene encodes a hydrophobic polypeptide of 444 ami no acids and is

the only structural gene required for C4-dicarboxylate transport in both

symbiotic and free-living rhizobip (2,5). The dctA promoter (5) is strongly

homologous to the consensus promoter -26 CTOGYAYR-N4-TTGCA -10 recognized by

the rpoN (ntrA) sigma factor (7), and recent results have shown that rpoN

© IRL Press Limited, Oxford, England. 7921

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Nucleic Acids Research

product (FpoN) 1B required for dctA expression (8). Other promoters known to

be recognised by RpoN such as qlnAp2 and nif promoters also require the

product of another regulatory gene, either the nitrogen regulator ntrC or the

nif-specific regulator nifA, for activation (reviewed in 7). The ntrC and

nifA products (NtrC and NifA respectively) are structurally similar, sharing

extensive regions of homology outside of their N-terminal domains that may

include a domain that interacts with RpoN (9,10). These observations suggest

that the dctA promoter may require an activator analogous to NtrC or NifA.

The regulation of dctA is complex. The dctA gene may be under negative

autogenous control, since a dctft-lacz fusion is expressed constitutively in

dctA mutants (5,11). In addition, two regulatory loci dctB and dctD that are

closely linked to dctA but probably transcribed divergently to dctA (1,2,5,

11), are required in addition to inducer and RpoN for dctA activation in free-

living rhizobia. The regulatory components of the dct regulon may also be

required for symbiotic nitrogen fixation, since nitrosoguanidine-induced dctB

mutants form nodules that fail to fix nitrogen (1,2). However only one out of

12 Tn5-induced dctB mutants formed nodules that fixed nitrogen at

significantly less than wild-type rates; the other Tn5-induced dctB mutants

and three Tn5-induced dctD mutants formed nodules that fixed nitrogen as well

as nodules formed by wild-type rhizobia (11). The reason for the disparity in

symbiotic phenotypes between the chemically-induced and transposon-induced

dctB mutants is not clear.

In this paper, we report the DNA sequence of dctB and dctD, and the

precise location and orientation of the Tn5 insertions. We report that dctB

product (DctB) is homologous over its C-terminal region to a variety of

proteins including ntrB product (NtrB) that may act as environmental sensors,

and that dctD product (DctD) is homologous over its N-terminal region with a

variety of proteins including NtrC that may act in conjunction with a sensor

component to activate transcription in response to environmental stimuli.

Furthermore DctD is strongly homologous in other regions to NtrC and NifA.

Based on these results, we propose that DctB is a receptor for C4-

dicarboxylates and transduces a signal across the membrane to DctD which then

acts in conjunction with RpoN to activate transcription of dctA. We also

propose a model to account for the symbiotic phenotypes of the regulatory

mutants.

AND METHODS

The region of DNA containing dctB and dctD was sequenced using specific

restriction fragments and nested sets of deletions generated using Bal 31

7922



nuclease (Fig. 1). Appropriate restriction fragments were subcloned from the

dct plasmids pPNlO3 and pRO.04 (2) into M13 mp8 and mp9 (12). The 1.2 kb

BamHI/EcoRI fragment and the 2.1 Sail fragment (Fig. 1) were subcloned into

pUC8 (13) and the resultant plasmids used as the sources of DNA for the

construction of Bal 31-generated deletions as described (8). Sequencing was

done by the dideoxy method using a 17-base universal primer and [ S)dATP as

label (14).

Sequence was also determined from the ends of several Tn5 insertions in

dctB and dctD. Recombinant plasmids that contained Tn5 insertions previously

used to generate dctB and dctD mutants by homogenotization (11), were digested

with EcoRI plus BamHI or BamHI alone, and the fragments cloned into pUC8.

Appropriate clones were screened for kanamycin resistance to determine the

orientation of the Tn5 insertion. The fragments were then cloned into M13 mp8

for sequence determination using a Tn5-specific primer as described (8). The

sequence generated also served to sequence across the EcoRI site in dctB (Fig.

1).

DNA sequences were compiled using the DBAUTO and DBOTIL programs (15), and

were analysed using programs from the University of Wisconsin (16). The

program FASTP (17) was used to screen the NBRF Protein Identification Resource

data base for sequences homologous to DctB or DctD. Pair-wise comparisons

between proteins were made with the ALIOJ program, using the mutation data

matrix and a gap penalty of 16 (18). The alignment scores given represent the

number of standard deviations separating the maximum score of the real

sequences from the mean of the maximum scores of 100 random permutations of

the two sequences (18).

dctA dctB dctD>133 3i>2006

; 7 1 s \ \ I I > K I I'v I Ik l v - „PP PBP X E XPPXPXKPS XP PS

1kb

Figure 1. Restriction map of the dct regulon from R. lequminosarum (2,11), andstrategy used to sequence the dctB and dctD genes. The heavy line indicatesthe region, the sequence of which is presented in Fig. 2. The sequence wasdetermined from restriction fragments (•—), deletions generated using Bal 31nuclease (i—-), and from the ends of Tn5 insertions (*—•). Restriction sites:B, BamHI; E, EcoRI; K, Kpnl; P, Pstl; S, Sail; X, Xhol.

7923



RESULTS AND DISCUSSION

Nucleotide sequences of dctB and dctD

In previcxis work, we defined the region of DNA containing dctB and dctD by

genetic complementation studies and transposon mutagenesis (2,5,11). We have

now sequenced thi6 region (Fig. 1), and also determined the orientation and

precise sites of several Tn5 insertions. The sequence data (Fig. 2) revealed

two open-reading frames (ORFs). The first ORF of 1866 nucleotides was

designated dctB since it corresponded precisely in location and orientation to

the region previously genetically defined to be dctB (2). Furthermore

sequence analysis showed that all Tn5 insertions that gave a DctB~ phenotype

(leaky growth on succinate) were located in this ORF. The translation

initiation site of dctB was arbitrarily assigned at the first of two possible

ATG codons which were separated by 12 bp. Neither ATG codon was preceded by a

good match to the consensus ribosome binding site defined for E. coli genes

(19). The first ATG codon of dctB was separated from that of dctA by 223 bp,

and the genes were transcribed divergently (data not shown). The second ORF

was designated dctD since the three Tn5 insertions that gave a DctD~ phenotype

(no growth on succinate) mapped within the ORF. The translation initiation

site of dctD was also assigned at the first of two potential ATG codons which

were separated by 3 codons, giving an ORF of 1344 nucleotides. The dctD

sequence was preceded by two potential ribosome binding sites, the first of

which (AAGG) overlapped dctB, and dctB and dctD were separated by 4

nucleotides (Fig. 2).

Eight out of the nine Tn5 insertions in dctB and dctD that were analyzed

were in the same orientation, with transcription of the nptll gene from left

to right with reference to Fig. 1. The insertion in strain 659, which is the

only Tn5-induced dctB or dctD mutant strain that formed Fix" nodules (11), was

the only insertion in the opposite orientation.

Structure of the dctB product

A search of the Protein Identification Resource protein data base using

the FASTP program (17) revealed that DctB shared homology over its C-terminal

region to the product of envZ, an E. coli gene involved in osmoregulation (20,

21). It has recently been reported that envZ product shares extensive

homology over its C-terminal 250 amino acids to analogous regions of the

products of the regulatory genes ntrB (22), cpxA (23), phoR (24), and virA

(25). Comparison of the C-terminal region of DctB with the other gene

products using the ALIGN program showed that several residues grouped in 5

regions were either invariant or substituted by a conservative change in all

six proteins. The product of a further gene phoM (26) was also found to

contain these homologous regions (Fig. 3).

7924



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Figure 2. Nucleotide sequence of dctB and dctp genes, and deduced amino-acidsequence of their products. Possible ribosome binding sites and ATGinitiation codons are overlined. The potential membrane-spanning regions ofdctB product are underlined. Sites of Th5 insertions are indicated byarrowheads together with the number of the induced mutant strain. Strains660, 533, 535, 543, 536 and 659 are dctB mutants, and strains 538, 663 and 537dctP mutants (11).

7925



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Figure 3. Alignment of C-terainal regions of products homologous to dctBproduct (RLDCTB), including the products of ntrB from Klebsiella pngiminniae(K 27) d d h l b l [ i ] ( P f 22) i Z (ECENVZp g p(KPNTRB; 27) and Bradyrhlzoblum spp. [Parasponia] (BPtfreB; 22), eiwZ (ECENVZ;

), p ( ; 23), phoR"TECPH0«; 24) and phoH (ECPHOM; 26) fromEscherlchia coli, and virA (AIVIRA; 25) from Agrobacterium tuaefaclens.•n H i i ~ T T t d f th i i l i t d t

;20), cpxA (BCCPXA;

, ( ; ) gimiH-ipi«<~flTTgnm»nt was prepared from the pair-wise alignments determined

using ALIGN by minimizing the number and location of gaps manually. Thisprocedure slightly reduced the alignment scores between any given pair ofsequences, but highlighted strongly conserved regions. Boxed residues areconserved, defined as more than 70% of amino acids belonging to one of thegroups P A G S T; H K R; Q N E D; I L V M F Y W C.

All the gene products homologous to DctB except NtrB contain two regions

of sufficient hydrophobicity to span the membrane, and virA and envZ products

have been shown to reside in the inner membrane (25,28). The hydropathy

profile of DctB (Fig. 4) showed that it also had two uncharged strongly

hydrophobic regions, one at residues 25 to 42 and one at residues 321 to 338.

The first potential membrane-spanning region was preceded by three arginine

residues, while the second region was preceded by two arginine residues and

7926



200 300 400 500 600

Figure 4. Hydropathy profile of dctB product at window-length 19, calculatedaccording to Kyte and Doolittle (29). Hydrophobic regions have positive valueand hydrophilic regions negative value.

followed by three arginine residues. Similar hydrophobic regions of 17 ormore residues surrounded by charged residues are known to be able to span thehydrophobic interior of the membrane (30,31). Hence the first hydrophobicregion of DctB may act as an uncleaved signal sequence, and the second regionas a "stop-transfer" or anchor sequence, as demonstrated for othertransmembrane proteins (30,31). Most of the N-terminal 320 amino acids ofDctB would then be periplasmically-located, while its conserved C-terminalregion would be located in the cytoplasm. The chemosensory receptor proteinsof E. coli have a similar structure (32) and the periplasmic domain has beenshown to bind the ligands (33,34). The binding of ligand is thought to causea signal to be transduced across the membrane to the cytoplasmic portion ofthe molecule. Similarly, binding of ligands to the periplasmic faces of theDctB homologues may cause activation of their conserved cytoplasmic regions.Structure of the dctD product

Like DctB, the regulatory proteins shown in Fig. 3 act in concert with asecond regulatory protein to regulate gene expression (reviewed in 35).Members of this second family which include ntrC (10,22), ompR (20), sfrA(36), phoB (37), virG (38,39) and "ORF2" (the gene downstream of phoM (26))products share extensive homology to each other and to the products of thecheY (40), cheB (40), spoOA (42) and spoOF (43) genes in their N termini ofabout 120 amino acids. DctD also shared this homology (Fig. 5). The N-terminal region is known to perform a regulatory function in cheB product(44). In the two-component systems, it may act as the receptor for signalstransmitted through the DctB homologues (22,35,38).

ompR, sfrA, phoB, virG and "0RF2" products share homology over theirentire length of about 240 amino acids, whereas NtrC diverges sharply afterthe H-terminal domain. However, the remainder of NtrC is highly homologous toanalogous portions of NifA, the positive regulator of rdf gene expression

7927



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rigure 5. Alignment of homologous regions of products conserved with dctpproduct (RLDCTD), including ntrC products from K. pneumoniae (KPNTOC; 9,10),Bradyrhlsoblum spp. [Parasponia] (BPNTRC; 22) and R. meliloti (RWWRC; 45);N-terminal regions of the products of ompR (ECOMPR; 20), sfrA (ECSFRA; 36),phoB (BCP90B; 37), cheY (BCCHEY, 41) andcheB (BCCHm; 4lTFrom Escnerlchiacoll (cheB and cheY from Salmonella typhimurium are similar (40)); and centraland C-teradnal regions of nifA products from KT pneirp̂ niae (KPNIFA; 9,10),B. meliloti (HMNIPA; 9) and R. legumunosarum TRLNIFA; 46). Alignments wereprepared as for Fig. 3. The potential nucleotide-binding site is underlined.

7928



Table 1. Pair-vdse alignment scores of the central regionsof products homologous to dctD product .

RLDCTDRPNTRCBPNTRCRMNTRCKPNIFARMNIFA

(145-389)(140-384)(140-383)(139-382)(212-455)(200-443)

KPNTRC43.9

BPNTRC4653

96

RMNTRC455883

864

KPNIFA40.546.244.954.3

RMNIFA38.347.345.842.654.8

RLNIFA43.644.348.449.559.566.2

Abbreviations are as in Figure 5.

(9,10). Analysis of DctD revealed that it was highly conserved with NtrC and

NifA proteins from several species in the regions where the NtrC and NifA

proteins are homologous to each other (Fig. 5). The regions of homology of

DctD with NtrC and NifA are consistent with the domain structure previously

proposed for the latter proteins (10). Hence DctD and NtrC share a common

N-tenninal domain linked by a hydrophilic segment (residues 120 to 142 of

DctD) to a conserved central region also found in NifA. The central region

may consist of more than one functional domain, since it contains segments of

strong homology separated by regions of lesser conservation. In particular,

the segment corresponding to residues 332 to 345 of DctD is hydrophilic but

poorly conserved in all proteins, suggesting it may be an inter-domain linker.

Following the conserved central region, and separated from it by a region of

variable length, all three products contain a helix-turn-helix motif

characteristic of a DNA-binding domain (ref. 10; asterisked in Fig. 5). The

linker between the conserved central and C-terminal domains is shortest in

DctD, accounting for most of the difference in size between DctD (448 amino

acids) and NtrC (469-480 amino acids) products.

The conserved central region and DNA-binding domain are likely to be

involved in functions common to NtrC, NifA and DctD. The central conserved

region of NtrC, NifA and DctD may be involved in interaction with RpoN or UNA

polymerase as all three proteins require RpoN as co-activator (8; reviewed in

7). Interestingly, the homology in the central region between NtrC proteins

from different species (mean score 65.2) and between NifA proteins from

different species (mean score 60.1) was greater than the homology between NtrC

and NifA proteins of the same species (mean score 44.4), or between NifA and

DctD from R. leguminosarum (score 43.6) (Table 1). This suggests that the

products diverged functionally before the species diverged evolutionarily.

Hence it seems likely that a progenitor gene encoding the conserved central

region has been adapted during evolution for use in several regulatory systems

that will require RpoN as co-activator. The consensus sequence characteristic

7929



dctD dctB „ _ _ _ dctA dctD dctB dcJA

Figure 6. Model for dctA activation. See text for details.

of RpoN-activated promoters has been observed in several structural genes

including xylA (47), carboxypeptidase G2 (48) and pilin genes (49) of

Pseudomonas species, and flagellin genes of Caulobacter crescentus (50). It

seems likely that each of these operons will require as co-activator a

regulatory gene product with hotnology to the central domain found in DctD,

NtrC and NifA.

The proposed DNk-binding domain may confer activator specificity on DctD,

NtrC and NifA, as NtrC and NifA each appear to recognize a specific nucleotide

sequence with dyad symmetry located at least 100 bp upstream of the promoters

they activate (51,52,53). The residues within the second helix of the helix-

turn-helix motif of the proteins, the helix that is likely to recognise the

specific binding site (54), are strongly conserved within the NtrC or NifA

proteins, but diverge between NtrC and NifA and with DctD. The dctA promoter

also contains regions of dyad symmetry upstream of the transcriptional

initiation site (unpublished data) and we are investigating whether these

regions are required for transcriptional activation by DctD.

DctD contains a potential nudeotlde-bindlng site in its central region

It has recently been shown that NtrB activates NtrC by phosphorylating it

(55). Examination of the sequence of NtrB or homologous regions of DctB did

not reveal hotnology to known protein kinases or ATP-binding sites. However a

potential ATP-blnding site was observed in strongly conserved regions of the

central portions of DctD, NtrC and NifA (underlined in Fig. 5). Walker et al

7930



(56) first observed two regions of hanology present in several nucleotide-

binding proteins, and these regions have been shown to form part of the ATP-

binding site of adenylate kinase (57). The first region in the central

portion of DctD etc., [G E S/T G S/T G K E] shows one mismatch to the

consensus [G X X G X G K S/T], while the second region ( A X G G T L F L D ]

contained two mismatches to the consensus [ G X X X L H y H y H y D ] , where Hy

represents an hydrophobic amino acid.

The presence of the potential ATP-binding site suggests that ATP may play

a role in the process of transcriptional activation mediated by DctD, NtrC and

NifA. In other proteins where it has been investigated, part of the consensus

ATP-binding site [G X X G X G K] is thought to form a flexible loop structure

that undergoes conformational change in response to either substrate binding,

as shown for adenylate kinase, or interaction with another protein or domain

(57). It has also been shown that mutations in a similar sequence of the ras

oncogene are responsible for the transforming ability of its product (58,59).

Mutagenesis of this region of DctD and its homologues should reveal whether

this sequence is involved in their activation in response to stimuli.

Model for dctA activation

We have shown that DctB and DctD are members of a conserved family of two-

component regulatory systems that respond to environmental stimuli (Figs 3 and

5; 22,35,38). These systems include E. coli genes responding to osmolarity

(envZ/oapR), nitrogen limitation (ntrB/htrC), phosphate limitation (phoiyphoB)

and toxic compounds (changes in membrane proteins controlled by cpxA/sfrA),

and genes controlling the virulence of Agrobacterium tumefaciens in response

to plant exudate (virA/virG). One component of each system is thought to act

as a sensor that transduces a signal to the second component which then

activates the response. The conservation in the C-terminal regions of the

sensors including DctB and the N-terminal regions of the activators including

DctD may reflect a common mechanism of signal transduction.

Given the horology between NtrB and DctB over their C-terminal regions and

the hanology of NtrC with DctD over their entire length, it is likely that the

Ntr and Dct systems will show common mechanisms in their mode of action. On

this basis, we propose that Dct regulation involves the following features

(Fig. 6): DctB and DctD are constitutively produced at low levels (11).

Binding of a C4-dicarboxylate to the periplasmic face of DctB causes an

allosteric change in the cytoplasmic portion of DctB, causing activation of

DctB. Activated DctB then interacts with the N-terminal region of DctD, the

interaction resulting in an allosteric or covalent modification of the

remaining portion of DctD. Activated DctD in conjunction with RpoN then

activates transcription of dctA. This activation may involve ATP, since DctD

7931



contains a possible ATP-binding site in its central region. Activated DctB

may be a protein kinase that phosphorylates DctD, since NtrB is a protein

kinase that activates NtrC by phosphorylating it (55). Alternatively,

activated DctB may induce a conformational change in DctD rendering it capable

of autophosphorylation.

One feature of dctA regulation not accounted for in the model is the

observation that a dctA-lacZ fusion is constitutively expressed in a dctA

mutant background (5,11). A similar phenomenon has been observed in another

two-component system that regulates a transport operon. Strains mutated

within the pst operon that encodes the high-affinity phosphate transport

system and is regulated by phoIVpnoB, express other phosphate-regulated

opercms constitutively (60). The three-prime gene of the pst operon phoU is

not required for phosphate transport, and it has been proposed that it

functions directly or indirectly with a cc— repressor transported by the pst

system to repress pho gene expression under conditions of phosphate excess

(61,62,63). However an alternative explanation also applicable to dctA is

that the structural gene products are in contact with the sensor component and

hold it in a particular mode in the absence of changes in stimuli. We are

isolating dctA dctB double mutants to test this idea.

The finding that most Tn5-induced dctB and dctD mutants form nodules that

fix nitrogen at wild-type rates suggested that dctA must be regulated

differently in symbiotic rhizobia (11). However dctB mutants induced by a

chemical autagen, and one Tn5-induced dctB mutant, strain 659, formed nodules

severely affected in their ability to fix nitrogen (1,11). The Tn5 insertion

in strain 659 was in the opposite orientation to those in the other dctB and

dctD mutants, we suggest that most Tn5-induced dctB mutants are polar on dctD

whereas the chemically-induced mutants and the Tn5 insertion in strain 659 are

apolar. Hence in these latter strains DctD will be present in inactive form.

Inactive NtrC is able to bind to the glnAp2 promoter but does not activate

transcription (51,52). Similarly, inactive DctD may bind to the dctA promoter

region and interfere with dctA activation by another system, possibly NifA, in

the symbiotic state.

Part of this work was supported by a grant from Hoechst AG to Massachusetts

General Hospital.

'Present address and address for correspondence: Biotechnica International Inc., 85 Bolton Street,Cambridge, MA 02140, USA4Present address: Department of Molecular and Cellular Biology, Pennsylvania State University,University Park, PA 16802, USA

7932



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7934


deduced products of c4-dicarboxylate transport regulatory genes of

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