FEMS Microbiology Letters 236 (2004) 21–31

www.fems-microbiology.org

Sinorhizobium medicae genes whose regulation involves the ActSand/or ActR signal transduction proteins

Beau J. Fenner a, Ravi P. Tiwari b,*, Wayne G. Reeve b, Michael J. Dilworth b,Andrew R. Glenn c

a Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore, Singapore 117604, Singaporeb Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology, Murdoch University, Perth, WA 6150, Australia

c Office of the Pro Vice Chancellor (Research), University of Tasmania, Hobart, Tasmania 7001, Australia

Received 27 January 2004; received in revised form 7 May 2004; accepted 12 May 2004

First published online 25 May 2004

Abstract

ActS–ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global

regulation in the a-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential

for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae. To do this,

random gusA fusions were created in S. medicae to follow gene transcription in an actS chromosomal knockout mutant containing

plasmid-borne actS. Plasmid borne actS was cured from the mutants and b-glucuronidase (GUS) activity compared between the

different genetic backgrounds. We detected actS-dependent regulation of the genes gst1 (detoxification), hyuA (hydantoin utiliza-

tion) and fixN2 (microaerobic respiration). We show that ActR is involved in regulating cbbS (CO2 fixation), narB (nitrate as-

similation) and required for low pH and microaerobic induction of the nitrogen fixation regulators fixK and nifA. In particular, we

demonstrate that the transcriptional activation of fixN2 is regulated by ActR through FixK.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: ActS; ActR; Two-component signal transduction; mTn5 mutagenesis; Low pH induction; General regulation; Redox sensing; Nitrogen

fixation; FixL; FixJ; FixK

1. Introduction

In recent years a new family of closely related two-

component signal transduction systems has been de-scribed in the a-subgroup of Proteobacteria. The first

described members were the RegB histidine kinase and

the RegA response regulator that are required for the

anaerobic induction of the photosynthetic apparatus of

the purple nonsulfur bacterium Rhodobacter capsulatus

[1,2]. RegBA act as a typical two-component system,

with the membrane-spanning RegB histidine kinase

phosphorylating RegA and allowing the activated reg-ulator to modulate transcription by interacting with

specific DNA targets [3–5]. RegBA homologs have been

* Corresponding author. Tel.: +618-9360-2202; fax: +618-9360-6486.

E-mail address: r.tiwari@murdoch.edu.au (R.P. Tiwari).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.05.016

discovered in other closely related anoxygenic photo-

trophs [6–9], plant-associated and soil bacteria [10–15]

and more recently in pathogens like Brucella melitensis

[16] and Mycobacterim tuberculosis [17].RegBA homologs regulate a diverse range of meta-

bolic processes including photosystem formation [9,18–

20], CO2 assimilation [21], nitrogen assimilation and

hydrogen uptake [22,23], respiration and electron

transport [24,25] and formaldehyde assimilation [26]. In

the root nodule bacteria, the RegSR system from

Bradyrhizobium japonicum is responsible for microaer-

obic induction of the nitrogen fixation regulator NifA,and regulation of CO2 assimilation [11,27]. RegBA

homologs from Rhodobacter sphaeroides regulate redox-

sensitive processes in response to perturbations in

reductant flow through the electron transport chain

[20,28,29]. DNA target sites for RegA homologs have

. Published by Elsevier B.V. All rights reserved.

22 B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31

been identified using footprinting studies and tran-

scriptional analysis [4,23,30–33].

Previously, we described the isolation, sequencing

and characterization of the RegBA homologs ActS and

ActR from the root nodule microsymbiont Sinorhizo-

bium medicae, both of which are essential for growth at

low pH [10]. ActS and ActR have been identified in

Rhizobium leguminosarum; knockout mutations in actS

and actR create acid sensitivity but impart slower

growth in neutral pH conditions [12].

The genetic targets for ActSR in Sinorhizobium have

not yet been elucidated. To initiate our investigation of

genetic regulation by the ActSR system we have created

Table 1

Bacterial strains and plasmids

Strain/plasmid Genotype/phenotype

E. coli

BW20767 RP4-2-tet:Mu-1kan::Tn7 integrant leu-63::IS10 recA1 c

DH5a supE44 DlacU169 (/80 lacZ DM15) hsdR17 recA1 end

Sinorhizobium strains

BF1212S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

BFL11 Cmr Gmr; fixL:pJQ200SK single crossover knockout

BFL11R Cmr Gmr Kmr; fixL:pJQ200SK single crossover knoc

GMI5630 Kmr Smr; D(nod fix)JB16 fixK1::Tn5 recA::Tn5-233 m

Rm1021 Smr derivative of Rm2011

RT295S Cmr Smr; actS::XSmr mutant of WSM419, acids

RTA6S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTA15S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTG47S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTL19S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTM11S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTN37S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

RTO33S Cmr Kmr Smr; mTn5-GNm mutant of RT295S

TG5-46 Cmr Kmr; actR:Tn5 mutant of WSM419, acids

WSM419 Cmr; wild type, acidt

Plasmids

pBF1212 14.1 kb HindIII fragment containing the BF1212S mT

pBFA15 20.5 kb HindIII fragment containing the RTA15S mT

pBFL11GS 1.1 kb ApaI–XbaI fixL fragment from pDP3 in pJQ2

pBFL19 11.6 kb HindIII fragment containing the RTL19S mT

pBFO33 6.6 kb EcoRI fragment containing the RTO33S mTn5

pCHK57 Tcr; pGD926 containing a 0.3 kb nifA fragment from

pCRS487 Apr Kmr; pUT::mTn5-GNm

pDP3 Gmr; pML133 containing a 2.9 kb blunted SnaI–Bam

pFUS1 Tcr; broad-host-range gusA transcriptional fusion vec

pGEM-7Zf()) Apr; cloning vector

pJJ5 Tcr; pIJ1363 containing a 0.8 kb fixJTK fragment fro

pJQ200SK Gmr; suicide delivery vector with sacB counter-selecta

pKW118 Apr; gusA translational fusion vector with trpA termi

pPFIX1 pFUS1 containing a 1.9-kb blunted BamHI–NotI fixN

pPH1JI Gmr Smr Spr; broad-host-range IncP plasmid

pREG118L pREG750S containing gusA from pKW118 cloned at

pREG118N pBRO1 containing the actR gusA fusion of pREG118

pREG750S S. medicae actR on a 0.8 kb PstI(blunted)–XbaI fragm

pRT546-20 S. medicae actSR on a 4.0 kb BglII–KpnI fragment in

pRT546-6 S. medicae actS on a 3.8 kb BglII fragment in pSW21

pRTA6 7.9 kb EcoRI fragment containing the RTA6S mTn5-

pRTG47 7.6 kb EcoRI fragment containing the RTG47S mTn

pRTM11 5.1 kb EcoRI fragment containing the RTM11S mTn

pRTN37 15.9 kb EcoRI fragment containing the RTN37S mTn

pSW172 Tcr; broad-host-range cloning vector

and screened a library of reporter gene fusions in

S. medicae. By identifying gene fusions regulated by

these proteins we provide insight into the types of pro-

cesses controlled by ActSR.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Strains and plasmids used in this work are listed in

Table 1. Strains of Sinorhizobium were grown at 28 �C in

TY medium or minimal salts medium [34] containing 20

Source/reference

reC510 hsdR17 endA1 zbf-5 gusA (DMluI)::pirþ thi [58]

A1 gyrA96 thi-1 relA1k� [59]

This work

mutant of WSM419 This work

kout mutant of TG5-46 This work

utant of Rm2011 [44]

[60]

This work

This work

This work

This work

This work

This work

This work

This work

[34]

[36]

n5-GNm fusion in pGEM-7Zf()) This work

n5-GNm fusion in pGEM-7Zf()) This work

00SK This work

n5-GNm fusion in pGEM-7Zf()) This work

-GNm fusion in pGEM-7Zf()) This work

S. meliloti Rm2011; nifA lacZ [45]

[36]

HI fixLJT fragment from Rm2011 [61]

tor [36]

Promega

m S. meliloti Rm2011; fixK-lacZ [44]

ble marker [62]

nator [38]

fragment from pBF1212; fixN-gusA This work

[63]

a SalI site in actR; (Plac)actR-gusA This work

L; (PactR)actR-gusA This work

ent in pSW172 This work

pMP220 [10]

3 [10]

GNm fusion in pGEM-7Zf()) This work

5-GNm fusion in pGEM-7Zf()) This work

5-GNm fusion in pGEM-7Zf()) This work

5-GNm fusion in pGEM-7Zf()) This work

[42]

B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31 23

mM DLDL-malate and 10 mM NH4Cl as the sole carbon

and nitrogen sources, respectively. Minimal media were

buffered with HEPES (pH 7.0) or MES (pH 5.7) at 40

mM. The final pH of liquid minimal media following

culturing did not vary by more than 0.2 units from theinitial pH. Cultures were routinely grown in 30 ml

McCartney bottles containing five ml of growth me-

dium. Where aerobic growth is specified, cultures were

grown in 100 ml Erlenmeyer flasks containing 10 ml of

growth medium. For microaerobic growth, cultures

were grown in seven ml Bijou bottles containing six ml

of growth medium. Both aerobic and microaerobic

cultures were shaken at 200 rpm on a gyratory platformshaker. Escherichia coli strains were grown at 37 �C in

Luria–Bertani medium [35]. When necessary, antibiotics

were added to cultures at the following concentrations

(lg mL�1): ampicillin, 50; chloramphenicol, 20; genta-

mycin, 10 (E. coli) or 40 (Sinorhizobium); kanamycin, 50;

streptomycin, 50 (E. coli) or 200 (Sinorhizobium); and

tetracycline, 12.5 (E. coli) or 20 (Sinorhizobium).

2.2. Genetic techniques and DNA analysis

Mutagenesis of S. medicae with mTn5-GNm was

performed as described previously [36]. Plasmids were

transferred from E. coli BW20767 hosts into S. medicae

recipients by conjugation [36]. Plasmid or genomic

DNA isolation, manipulation, modification and trans-

pH 7.0 pH 5.7

low pH repressed

low pH induced

no pH effect

curinpPH

p

actS actR

pRT546-6

mTn5-GNm

actS

pREG7

mTn5-G

pRT54

mTn5-G

mutagenesis with mTn5-GNm1

Sinorhizobium medicae RT295S (pRT546-6)

actS actR

pRT546-6

genome

actS

Fig. 1. Schematic diagram of the genetic scree

formation techniques used have been described else-

where [37]. Kanamycin resistant EcoRI fragments were

cloned from each mutant and the DNA sequence

flanking the site of a mTn5-GNm insertion was gener-

ated using the WIL3 [38] and TAC-105F primers [36]and queried against the Sinorhizobium meliloti Rm1021

genomic database [39] using the BLAST algorithm.

2.3. Plasmid mobilization and curing

Plasmids were transferred into Sinorhizobium strains

by conjugation as described previously [40]. Transfer of

pPH1JI and simultaneous loss of pRT546-6 was con-firmed by replica plating and isolating the GmR and TcS

clones (Fig. 1 step 2). Transfer of pMP220, pSW213

(and derivatives) with concomitant loss of pPH1J1 was

confirmed by replica plating and selecting for GmS and

TcR clones (Fig. 1 step 3).

2.4. Enzyme assays

For quantitative measurement of b-glucuronidase(GUS) activity, strains of Sinorhizobium were inoculated

into liquid minimal medium and grown to late log phase

(A600 0.6–0.8). Cells were pelleted, resuspended in saline,

and inoculated into fresh minimal medium at an A600 of

either 0.005–0.01 for pH 7.0 aerobic, 0.1 for pH 7.0

microaerobic, or 0.2 for pH 5.7 to compensate for the

g with1JI

2

3lasmid replacement &quantitative screening

actS actR

pPH1JI

mTn5-GNm

actS+ actS–

actS actR

50S

Nm

Plac

actS actR

pSW172

mTn5-GNm

actR

actS actR

6-20

Nm

actS

actS actR

pMP220

mTn5-GNm

actR

Plac

n used to identify ActR regulated genes.

24 B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31

differences in growth rate at neutral pH, microaerobic

and low pH environments. Cultures were then harvested

at A600 0.2–0.6 after 20–25 h of growth. GUS activity

was measured as described previously [40]. b-Galacto-

sidase activities were determined in essentially the sameway except that reactions were carried out in modified

Z-buffer (0.1 M sodium phosphate, pH 7.0, 10 mM KCl,

1 mM MgSO4, 5 mM DTT) using o-nitrophenyl-b-DD-galactopyranoside at a final concentration of 0.4 mg

mL�1 as the substrate. Endogenous b-galactosidase ac-

tivity in Sinorhizobium cells was inactivated as described

previously [41]. Protein concentrations in cell prepara-

tions were determined with a Bio-Rad protein assay kitusing BSA as the protein standard.

3. Results

3.1. Genetic screen to identify ActS-responsive genes

The ActSR orthologs are involved in global gene reg-ulation in the a-proteobacteria. A genetic screen

was conducted to identify the types of genes regulated in

S. medicae through ActS (histidine kinase ‘sensor’ pro-

tein). The first step in this approach was to construct a

library of mTn5-GNm induced mutants of RT295S

(actS::XSmr) containing pRT546-6 (an actS-comple-

menting plasmid), thereby generating a pool of strains

containing transcriptional fusions to gusA (Fig. 1, step 1).This approach enabled us to later eliminate actS by

plasmid curing (Fig. 1, step 2). Approximately 5000

mutants were patched onto pH 7.0 or 5.7 minimal me-

dium plates containing the chromogenic GUS substrate

X-Glc and inspected for differences in color development

to identify pH-responsive gusA transcriptional fusions.

Transconjugants showing differences in the intensity of

blue color at low and neutral pH buffered plates wereselected for further screening. The selected strains were

Table 2

Effect of multicopy actSR or overexpressed actR on expression of gusA tran

Mutant b-Glucuronidase specific activity (nmol pNP min�1 [mg

Comparison 1 Difference (fold)b

pMP220a

(control)

pRT546-20

(multicopy actSR)

RTA6S 17.0� 5.1 4.4� 0.9 # 3.9�RTA15S 41.5� 0.4 35.8� 4.9

RTG47S 58.9� 7.7 38.1� 2.1

RTL19S 79.2� 4.5 329� 28.5 " 4.2�RTM11S 20.1� 1.4 8.5� 0.1 # 2.4�RTN37S 127� 13.6 238� 1.2 " 1.9

RTO33S 352� 69.3 20.7� 0.1 # 17�BF1212S 3.3� 1.0 12.3� 3.0 " 3.7�a The low copy number parent plasmids pMP220 and pSW172 are maintabGUS activity was either induced ð"Þ or repressed ð#Þ in mutants contain

pMP220 or pSW172, respectively. Values shown are the average of at least tw

first cured of the existing actS plasmid (pRT546-6) by

mobilizing the incompatible plasmid pPH1J1. Another

plasmid construct (pRT546-20, pMP220, pREG750S or

pSW172) was then reintroduced using conjugational

transfer and transconjugants devoid of pPH1J1 wereisolated. To determine if ActS regulated the transcription

of selected fusions, GUS activity was compared between

isogenic strains either containing, or devoid of, actS

(Fig. 1, step 3). Quantitation of GUS revealed that the

expression of six transcriptional fusions differed by �2-

fold or more between these two genetic backgrounds

(Table 2). Expression of GUS decreased in three mutants

(RTA6S, RTM11S, RTO33S) but increased in anotherthree (RTL19S, RTN37S, BF1212S) in the presence of

multicopy actSR (Table 2). Transcriptional fusions

whose expression did not differ by more than 20% be-

tween the different actS genetic backgrounds were con-

sidered as being actS unresponsive (data not shown).

3.2. Isolation of ActR regulated genes

Previously, we showed that multiple copies of actR

rescue an actS mutant from acid-sensitivity [10] sug-

gesting that the presence of multiple copies of actR

overrides the requirement for activation of ActR by

ActS. We therefore reasoned that additional genetic

targets could be identified from the pool of pH-re-

sponsive gusA fusions by screening cells in the presence

and absence of an excess dosage of ActR. Mobilizationof pREG750S into S. medicae mutants allowed for high-

level expression of actR from Plac in pSW172 [42] since

the Plac promoter in this plasmid yields approximately

13 times more ActR than the native PactR promoter

(data not shown). By comparing GUS activities between

isogenic strains containing pREG750S or pSW172, two

mutants (RTA15S and RTG47S) were identified that

contained fusions that were down-regulated by overex-pressed actR (Table 2). Comparisons of GUS activities

scriptional fusions in actS mTn5-GNm mutants

protein]�1)

Comparison 2 Difference (fold)b

pSW172a

(control)

pREG750S

(multicopy Plac-actR)

15.7� 1.6 14.7� 0.4

57.7� 8.5 21.6� 0.1 # 2.7�49.1� 6.1 13.7� 1.6 # 3.6�66.2� 6.7 34.3� 0.2 # 1.9�23.1� 5.7 19.1� 0.2

138� 2.5 169� 5.1

281� 39.5 552� 35.8 " 2.0�3.4� 0.7 3.2� 0.2

ined at 5–10 copies per cell [42,64].

ing pRT546-20 or pREG750S in comparison to the parent plasmids

o independent cultures grown at pH 7.0 and assayed in quadruplicate.

B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31 25

for the six initially identified mutants revealed that in the

case of RTL19S and RTO33S, the regulation of GUS

activity was dependent on both the presence of actS and

the dosage of actR (Table 2).

3.3. Effect of pH on actS or actR responsive fusions

Comparisons of GUS activity between mTn5 mu-

tants complemented with plasmid-borne actSR grown at

either pH 7.0 or 5.7 showed either no effect on expres-

sion or a difference of up to 7-fold induction (Fig. 2).

Although initial selection of mutants was based on dif-

ferences in colour development on neutral and low pHmedia plates containing the substrate X-Glc, surpris-

ingly two of the mutants RTM11S and RTN37S did not

Fig. 2. Low-pH regulation of ActSR-regulated gusA fusions in mTn5-

GNm mutants. GUS activities of mTn5-GNm mutants containing

either pRT546-20 (multicopy-actSR; filled bars) or pMP220 in an actS

background (unfilled bars) and grown at pH 7.0 have been compared

with mutants grown at pH 5.7. Values of approximately 1 (indicated

by a dotted line) represent no significant difference in GUS activity

between the different pH values. GUS activities (standard error <15%)

were measured from at least two independent cultures assayed in

quadruplicate.

Table 3

Sequence homologies of DNA regions flanking the sites of mTn5-GNm inse

Mutant Homologous gene(s) Putative gene function

RTA6Sb SMc04059/glgP Hypothetical protein/glycogen phosp

RTA15S cbbS Ribulose-1,5-bisphosphate carboxyla

subunit

RTG47S narB Assimilatory nitrate reductase large

RTL19Sb SMc00795 Hypothetical protein

RTM11S hyuA Hydantoin utilization protein

RTN37Sb gst1 Glutathione S-transferase

RTO33S SMc00888 PAS two-component receiver protein

BF1212S fixN2O2 cbb3 Cytochrome oxidase componen

Sequences were compared to the S. meliloti Rm1021 genomic database [a Significance of homology refers to the probability that such a match woubThe mTn5-GNm insertion in RTN37S is 10 bp downstream from the puta

the putative stop codon of SMc4059 and 121 bp upstream of the putative s

putative start codon of SMc00795.

show pH dependent regulation when GUS activity was

quantified in broth cultures. It demonstrates the im-

portance of GUS quantification in broth cultures to

establish real differences in gene expression.

When the mTn5 mutants devoid of plasmid-borneactSR were grown in the same conditions, a similar level

of pH response was observed with all mutants with the

exception of BF1212S. In this strain complemented with

plasmid-borne actSR the gusA fusion was induced ap-

proximately 4-fold at low pH (Fig. 2) with the activity

rising from approximately 14 to 60 nmol pNP min�1

(mg protein)�1 at pH 7.0 versus pH 5.7 respectively.

However, in an actS background, GUS activity at bothpH 7.0 and 5.7 fell to approximately 3.0 nmol pNP

min�1 (mg protein)�1.

3.4. Sequence analysis of DNA flanking mTn5-GNm

insertions

Southern hybridization experiments revealed that

each of the selected mutants was the result of a singlegenomic mTn5-GNm insertion event (data not shown).

Sequence comparison with the homologous S. meliloti

Rm1021 sequence showed that insertions occurred in

eight unique positions (Table 3) with four located in

intergenic regions and four intragenic. The mTn5-GNm

insertion in RTN37S occurred downstream from a

putative stop codon of a gene encoding glutathione S-

transferase. The insertion in RTL19S was 47 bp up-stream of the putative start codon of a gene encoding a

hypothetical protein showing similarity to OsmB (os-

motically induced lipoprotein B precursor) from E. coli.

The mutant RTA6S contains mTn5-GNm between the

stop codon of a gene encoding a hypothetical protein

and the start codon of glgP, encoding glycogen phos-

phorylase. The BF1212S insertion occurred within a 13

bp intergenic sequence between the fixN2 and fixO2

genes. Intragenic insertions occurred in the mutants

RTA15S, RTM11S, RTO33S and RTG47S, disrupting

rtion in ActSR-responsive mutants

Genome location in Rm1021 Significance of homologya

horylase Chromosome 9� 10�37/6� 10�67

se small pSymB 0

subunit pSymB 0

Chromosome 4� 10�8

pSymB 0

Chromosome 2� 10�69

Chromosome 6� 10�81

ts pSymA 0

39].

ld occur by chance as determined by the BLASTX algorithm [65].

tive stop codon of gst1; the insertion in RTA6S is 77 bp downstream of

tart codon of glgP; the insertion of RTL19S is 47 bp upstream of the

26 B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31

genes encoding the small subunit of RuBisCO, a hy-

dantoin utilization protein, a PAS protein containing a

two-component receiver domain and the large subunit

of an assimilatory nitrate reductase (Table 3). Sequence

comparisons made with the S. meliloti Rm1021 genomicdatabase showed that the ActSR-regulated genes are

distributed throughout each of the three replicons of S.

meliloti (chromosome, pSymA, and pSymB).

3.5. ActSR and FixLJ coordinately regulate fixN2

expression in S. medicae

Expression of fixN in S. meliloti is dependent on atleast two regulators; the FixJ response regulator, which

is activated by the FixL histidine kinase, and the FNR-

family FixK regulator [43]. Though a functional copy of

actS is clearly required for the low pH induction of

fixN2 in S. medicaeWSM419, this effect could have been

mediated through these regulators. We therefore created

a single crossover fixL knockout mutation in parental

and actR genetic backgrounds. A fixN2-gusA tran-scriptional reporter plasmid, pPFIX1, was then mobi-

lized into both of these strains and also into parental,

actS and actR strains. In the wild type background,

expression of fixN2 was induced 16-fold by microaero-

biosis at pH 7.0 and 2-fold by low pH (pH 5.7) under

aerobic conditions (Table 4). In actS and actR back-

grounds, expression of fixN2 was reduced to approxi-

mately half that observed in the wild type under bothaerobic and microaerobic conditions, while the low pH

induction of fixN2 was essentially abolished. The mi-

croaerobic induction of fixN2 at low pH was similar to

that observed at neutral pH, with GUS activities for

microaerobically grown wild type cells of 605� 87.2 and

490� 67.1 nmol pNP min�1 (mg protein)�1 at pH 7.0

and 5.7, respectively. Inactivation of fixL reduced the

microaerobic induction of fixN2 from 16-fold to 2-fold,but did not affect the observed low pH induction (Table

Table 4

Regulation of fixN 2 by ActSR, FixLJ and FixK. GUS assays were performed

gusA reporter plasmid pPFIX1

b-Glucuronidase specific activity

Strain and genotype pH 7.0 aerobic

S. medicae strains

WSM419 (wild type) 36.6� 0.5

RT295S (actS) 16.3� 3.2

TG5-46 (actR) 19.5� 0.9

BFL11 (fixL) 32.1� 1.9

BFL11R (actR-fixL) 15.1� 1.2

S. meliloti strains

Rm1021 (wild type) 38.3� 1.9

GMI5630 (fixK1-fixK2) 30.4� 1.3

Values shown are the average of at least two independent cultures assay

4). In an actR-fixL double mutant the 2-fold microaer-

obic and low pH induction of fixN2 was undetectable,

indicating that both ActR and FixL positively regulate

fixN2 during microaerobiosis but that only ActR directs

a low pH transcriptional response.

3.6. FixK is required for low pH induction of fixN2 in

S. lmeliloti

Microaerobic activation of fixN by the FixLJ system

is channelled through the FixK regulator in S. meliloti

[44]. The possibility of ActR activating fixN expression

through FixK in S. meliloti was explored by examiningexpression of the fixN2-gusA fusion on plasmid pPFIX1

in the mutant GMI5630 [44] and the wild type S. meliloti

1021; GMI5630 does not carry a functional fixK gene as

only two copies of fixK are present in the S. meliloti 1021

genome [39]. The expression of fixN2 fusion derived

from S. medicae was increased 3-fold in strain S. meliloti

1021 by low pH, whereas in the S. meliloti fixK mutant

there was no significant change at low pH (Table 4).

3.7. fixK is regulated through ActR in S. medicae

It was reasoned that if FixK is required for both the

microaerobic and low pH induction of fixN2, then ActR

should regulate the expression of fixK in a microaerobic

and low pH-dependent manner. This possibility was

investigated by assaying S. medicae parental and actR

strains carrying the S. meliloti fixK-lacZ translational

fusion on the plasmid pJJ5 [44]. In both strains of S.

medicae WSM419 the fixK fusion was induced 30- to 40-

fold during microaerobic growth, though the overall

level of expression in the actR strain was only half that

of the parental strain. The fusion was also induced 13-

fold in the parental strain when exposed to acidity, but

only 3-fold in the actR strain exposed to the same con-ditions (Fig. 3).

on the indicated strains of S. medicae and S. meliloti carrying the fixN2

(nmol pNP�1 min�1 [mg protein]�1)

pH 7.0 microaerobic pH 5.7 aerobic

605� 87.2 66.8� 5.4

366� 20.4 17.8� 0.2

358� 15.0 26.8� 2.8

60.3� 6.1 64.9� 1.8

17.3� 0.4 14.5� 1.1

409� 36.2 106� 9.4

34.3� 1.6 37.7� 2.9

ed at quadruplicate.

Fig. 3. Microaerobic and low pH activation of fixK by ActR. Ex-

pression of the fixK-lacZ translational fusion carried on plasmid pJJ5

[44] was determined in cultures grown aerobically or microaerobically

at pH 7.0 and pH 5.7 in WSM419 (wild type; filled bars) and TG5-46

(actR; unfilled bars). b-Galactosidase activities are expressed as nmol

ONP min�1 (mg protein)�1 and were measured from at least two in-

dependent cultures assayed in quadruplicate. Error bars indicate the

standard error of the mean.

B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31 27

3.8. ActR regulates nifA in S. medicae during microaer-

obic and low pH growth

ActR homologs from Rhodobacter spp. and B. ja-

ponicum activate nifA, encoding the nitrogen fixation

regulator NifA, in response to microaerobiosis [11,23].

Like fixK, the nifA gene of S. meliloti is also positively

regulated by FixJ in microoxic conditions [45]. To de-

termine if ActR regulates nifA in S. medicae, we used the

500

1000

1500

2000

2500

0pH 7.0aerobic

pH 7.0micro-aerobic

pH 5.7aerobic

3000

Fig. 4. Microaerobic and low pH activation of nifA by ActR. Ex-

pression of the nifA–lacZ translational fusion carried on plasmid

pCHK57 [45] was determined in cultures grown aerobically or mi-

croaerobically at pH 7.0 and pH 5.7 in WSM419 (wild type; filled bars)

and TG5-46 (actR; unfilled bars). b-Galactosidase activities are ex-

pressed as nmol ONP min�1 (mg protein)�1 and were measured from

at least two independent cultures assayed in quadruplicate. Error bars

indicate the standard error of the mean.

plasmid pCHK57 which carries a nifA lacZ fusion de-

rived from S. meliloti [45]. In wild type S. medicae, this

fusion was induced 6-fold by low oxygen and 2-fold by

low pH. In the actR mutant, nifA expression still re-

sponded to low oxygen and low pH, though the induc-tion in both cases was reduced by 30–40% (Fig. 4).

4. Discussion

To initiate our investigation of the ActSR signal

transduction system of S. medicae we have used mini-

transposon mutagenesis to identify genes whose tran-scription involves ActSR. The histidine kinase ActS

mediated negative or positive regulation of the identified

loci under aerobic conditions at pH 7.0. The transcrip-

tion of two genes, SMc00795 (in RTL19S) and SMc00888

(in RTO33S), was altered by both the presence of ActS

and the dosage of ActR. SMc00795, for example, was

induced 4-fold by multicopy ActS, but repressed 2-fold in

the presence of overexpressed ActR (Table 2). RegAfrom R. capsulatus also exhibits this dual negative and

positive regulatory function on its target promoters, and

this switch apparently depends on the phosphorylation

state of RegA, mediated through the RegB histidine ki-

nase [29,46,47]. If the same situation occurs in S. medicae,

phosphorylated ActR would therefore activate tran-

scription whereas unphosphorylated ActR would repress

transcription in the case of SMc00795.Comparisons of DNA sequences near the minitrans-

poson insertions with the S. meliloti Rm1021 genome

sequence revealed putative functions for six of the eight

interrupted loci (Table 3). The cbbS gene, encoding the

small subunit of RuBisCO, was repressed by ActR in the

absence of ActS. Similarly, cbb promoter activity in B.

japonicum is repressed by RegR, though this repression

only occurs in cbb-deficient mutants [48]. In addition toCO2 assimilation, the CBB reductive pentose phosphate

pathway also functions to dissipate excess reductant in

Rhodobacter spp. RegBA appear to coordinately regu-

late this and other reductant-dissipating pathways to

maintain cellular redox balance [8,49]. Other genes

regulated by ActSR, including narB, gst1, SMc00888

and hyuA, suggest that ActSR regulates diverse meta-

bolic processes such as nitrate assimilation, detoxifica-tion, additional signal transduction pathways and

hydantoin metabolism, though the physiological rele-

vance of this regulation remains unknown.

Of particular importance was the finding that ActS is

involved in responses to both microaerobic and acidic

conditions. Previous studies on ActS homologs from

other species have focused on reduced oxygen tension

as an activating signal for this system, though our dataclearly demonstrate that ActS can also transduce a low

pH signal. Current data suggest that in R. sphaeroides

Fig. 5. Model comparing low oxygen and/or low pH regulation of fix and nif genes by the FixLJ and ActSR (RegSR) two-component systems of

(a) Sinorhizobium and (b) B. japonicum. During growth under low oxygen and/or low pH conditions, the membrane-bound histidine kinases FixL and

ActS/RegS activate their respective response regulator partners FixJ and ActR/RegR. The active response regulators then regulate the expression

target genes, presumably by binding to upstream promoter elements. The symbol � has been used to indicate either direct or indirect involvement.

28 B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31

the flow of reductant through the microaerobically

inducible cbb3 cytochrome oxidase generates an inhib-

itory signal under aerobic conditions to maintain the

ActS homolog, PrrB, in the phosphatase-dominant

state, thus preventing activation of the PrrA response

regulator [20,28,50,51]. Similarly, oxidized electroncarriers generated by terminal oxidases inhibit the ki-

nase activity of the ArcB histidine kinase of E. coli,

preventing phosphorylation of the global response

regulator ArcA under aerobic conditions [52]. The

finding that both ActR (this study) and ArcA [53] can

transduce a low pH signal in addition to a microaerobic

signal suggests that low pH stimulates both ArcB and

the ActS homologs. It is possible that at low pH theproton pumping activity [54–56] of the inhibiting ter-

minal oxidases is retarded, leading to a reduced re-

ductant flow and therefore activation of the histidine

kinase. Interestingly, defects in cytochrome bd oxidase

expression caused by the interruption of cydB in Bru-

cella abortus lead to extreme acid sensitivity [57]. This

sensitivity is postulated to occur due to an accumula-

tion of oxidative radicals at low pH that overruns thecell’s protective enzymes such as superoxide dismutase

and catalase [57]. In light of these findings, future work

will focus on the potential involvement of electron

transport components such as cbb3 oxidase in the acid

tolerance of S. medicae.

To our knowledge this is the first report of a second

two-component system, in addition to FixLJ, that mod-

ulates expression of the fixK-fixNOQP and nifA genes inSinorhizobium. Unlike Sinorhizobium, the microaerobic

induction of nifA in B. japonicum is independent of

FixLJ, but absolutely requires the ActSR homologs,

RegSR [11]. In B. japonicum, therefore, an intact RegSR

system is essential for symbiotic N2fixation [11], while the

FixLJ system is also essential through its induction of the

FixNOQP system through FixK2 (Fig. 5(b)).

The situation is markedly different in S. medicae,

where the actR mutant, TG5-46, fixes nitrogen symbi-otically at rates comparable to the wild type (unpub-

lished observations). To integrate our observations of

the regulation of fixK-fixNOQP and nifA by ActSR and

FixLJ in S. medicae we propose a working model of

this regulatory circuit, illustrated in Fig. 5(a). Low O2

tension and low pH signals are transmitted via ActS

and ActR to positively affect transcription of fixK, fixN

and nifA, though it is unclear whether these effects ofActR are directly on the relevant promoters or indirect.

Though an intact ActSR system is required for maxi-

mum microaerobic induction of nifA, microaerobic in-

duction of nifA by the FixLJ system in S. medicae is

apparently fully sufficient for symbiotic nitrogen fixa-

tion even in the absence of ActSR, thus explaining the

difference in symbiotic behaviour of mutants in

RegSR and ActSR in B. japonicum and S. medicae,respectively.

Acknowledgements

We thank Jacques Batut, Daniel Kahn, Gary Ditta

and Stephen Winans for supplying strains and plasmids

used in this study. B. Fenner was supported by anAustralian Postgraduate Award. Additional support

was provided by the Australian Research Council.

B.J. Fenner et al. / FEMS Microbiology Letters 236 (2004) 21–31 29

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