Role of TNF in the Altered Interaction of Dormant Mycobacterium tuberculosis with Host Macrophages

ORIGINAL ARTICLE

Characterization of an rpoN mutant of Mesorhizobium ciceriU.S. Gautam1, A. Jajoo2, A. Singh1, P.K. Chakrabartty3 and S.K. Das4

1 National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, Pusa, New Delhi, India

2 School of Life Sciences, Devi Ahilya Vishwavidyalaya, Indore, India

3 Madhyamgram Experimental Farm and Department of Microbiology, Bose Institute, Kolkata, India

4 Institute of Life Sciences, Department of Biotechnology, Nalco Square, Bhubaneswar, India

Introduction

The members of the soil bacteria Rhizobiaceae infect the

legume roots and incite the formation of nodules. Meso-

rhizobium ciceri induces the formation of nitrogen-fixing

nodules on the roots of chickpea (Cicer arietinum).

Within the infected cells of the nodules, the bacteria are

enveloped in a membrane of plant origin, called the

peribacteroid membrane (PBM), and divide and differ-

entiate into nitrogen-fixing bacteroids. In the nitrogen-

fixation process of rhizobium–legume symbiosis, a large

amount of energy is required for the enzyme nitrogenase

to reduce atmospheric nitrogen to ammonia. The energy

becomes available from the oxidation of reduced carbon

supplied by the host to the bacteroid. C4-dicarboxylates

are the major carbon source taken up by bacteroids and

dicarboxylate transport (Dct) system is essential for an

effective symbiosis (Ronson et al. 1981). Strains of

Rhizobium leguminosarum bv. viciae, Rhizobium legumi-

nosarum bv. trifolii, Sinorhizobium meliloti with nonfunc-

tional Dct system formed ineffective nodules, and their

ability to fix nitrogen was dependent on and influenced

by functional dct system (Ronson et al. 1981; Finan et

al. 1983; Bolton et al. 1986). Genetic analyses of Dct)

mutants showed that the gene for rhizobial C4-dicarb-

oxylate permease (dctA) is regulated by the alternative

sigma factor, RpoN, and the two-component regulatory

system DctB ⁄ D (Jiang et al. 1989).

The alternative sigma factor, RpoN, also known as r54,

is encoded by the gene rpoN (ntrA, glnF). RpoN is essen-

tial for transcription initiation by RNA polymerase at

promoters characterized by an invariant GG doublet at 24

and a GC doublet at 12 upstream of the transcriptional

start site (Dixon 1987). The gene rpoN works in

Keywords

Cicer arietinum (L), dicarboxylate transport,

Mesorhizobium ciceri, nitrogen fixation, rpoN

gene, transposon mutagenesis.

Correspondence

Subrata K. Das, Institute of Life Sciences,

Department of Biotechnology, Nalco Square,

Bhubaneswar – 751 023, India.

E-mail: [email protected]

2006 ⁄ 1736: received 11 December 2006,

revised 13 March 2007 and accepted 21

March 2007

doi:10.1111/j.1365-2672.2007.03432.x

Abstract

Aims: To study the genetic basis of C4-dicarboxylate transport (Dct) in relation

to symbiotic nitrogen fixation in Mesorhizobium ciceri.

Methods and Results: A Tn5-induced mutant strain (TL16) of M. ciceri, unable

to grow on C4-dicarboxylates, was isolated from the wild-type strain TAL 620.

The mutant lacked activities of the enzymes, which use C4-dicarboxylates as

substrate. The sequencing of the 3Æ2kb EcoRI fragment, which was the site of

Tn5 insertion, revealed three complete and two partial open reading frames. In

the mutant, Tn5 interrupted the rpoN gene, of which only one copy was there.

Complementation and biochemical studies suggest that the M. ciceri rpoN activ-

ity is required for C4-Dct, maturation of bacteroids and symbiotic nitrogen fix-

ation. The fine structure of the ineffective nodules produced by TL16 on Cicer

arietinum L changed in comparison with those produced by the wild type.

Conclusions: The mutant strain TL16 suffered a disruption in the rpoN gene.

Only one copy of rpoN gene is present in M. ciceri. The mutation abolishes

Dct activity. It additionally abolishes the symbiotic nitrogen fixation activity of

the bacteroids in the nodules.

Significance and Impact of the Study: This first document in M. ciceri shows

that a functional rpoN gene is essential for the transport of dicarboxylic acids

and symbiotic nitrogen fixation.

Journal of Applied Microbiology ISSN 1364-5072

1798 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1798–1807

ª 2007 The Authors

conjunction with the NtrC class of transcriptional activa-

tors to control the expression of diverse genes, including

those responsible for nitrogen fixation, Dct, assimilation

and dissimilation of nitrate and catabolism of aromatic

compounds (Kustu et al. 1989; Merrick 1993). The rpoN

has been reported from the symbiotically nitrogen-fixing

bacteria Rhizobium etli (Michiels et al. 1998b), Sinorhizo-

bium meliloti (Ronson et al. 1987), Rhizobium sp. strain

NGR234 (Van Slooten et al. 1990) and Bradyrhizobium

japonicum (Kullik et al. 1991) for its role in nitrogen

metabolism. In S. meliloti, the rpoN locus has been

mutated and sequenced (Ronson et al. 1987). The mutant

formed Fix- nodules on alfalfa and failed to grow on

C4-dicarboxylates (Albright et al. 1989), while rpoN

mutant of Rhizobium sp. strain NGR234 showed pleio-

trophic phenotype, including an effect on nodulation

gene expression (Van Slooten et al. 1990). In R. etli, two

rpoN genes encoding the r54 have been characterized and

shown to be differentially regulated (Michiels et al.

1998a). During free living growth, rpoN1 is required for

growth on several nitrogen and carbon sources. There is a

severe decrease in nitrogen fixation after inactivation of

rpoN2 (Michiels et al. 1998a), indicating the essential role

of this gene in nitrogen fixation by bacteroids. To date,

no information, whatsoever, is available on the rpoN regu-

lon of M. ciceri. The genetic basis of C4-Dct, in relation

to formation of nodules and nitrogen fixation activity in

this species, has not been studied.

In the present study, we report the isolation of a Tn5-

induced mutant of M. ciceri, strain TL16. The mutant is

phenotypically nonfunctional in Dct and forms ineffective

nodules. Cloning of the mutant gene and analysis of the

nucleotide sequence of the gene provide evidence that, in

the mutant strain TL16, the rpoN gene is disrupted by

Tn5. We conclude that rpoN-encoded alternative sigma

factor is required for symbiotic functions: nodule organo-

genesis, and nodule functions like C4-Dct and nitrogen

fixation in M. ciceri. To the best of our knowledge, this is

the first report suggesting a role of rpoN in symbiotic

functions, including transport of dicarboxylate and

synthesis of the enzymes which use C4-dicarboxylate as

substrate in M. ciceri.

Materials and methods

Bacterial strains, plasmids and media

Bacterial strains and plasmids used in this study are listed

in Table 1. Mesorhizobium ciceri was grown in yeast

extract-mannitol (YM) medium (Vincent 1970) or in rhiz-

obium minimal medium containing: (g l)1) K2HPO4, 2Æ0;

KH2PO4, 1Æ5; NaCl, 0Æ15; NH4Cl, 0Æ5; MgSO4.7H2O, 0Æ5;

Table 1 Bacterial strains and plasmids used

Bacterial strains Relevant phenotype* Reference

Escherichia coli S17Æ1 Pro, hsdR, recA [RP4.2 (Tc::Mu) (Km::Tn7)] Simon et al. (1983)

DH5a E. coli host strain, endA1 hsdR17 supE44

thi-1 recA1 gyrA96 relA1 D (argF-lac ZYA)

Bethesda Research

Laboratories, Inc.

HB101 F-hsdS20(rB- mB

-) recA13 ara-14 proA2 LacY1

galK2 rpsL20 xyl-5 mtl-1 supE44k-

Promega

Mesorhizobium ciceri

TAL620 (wild type)

Nxr, Apr, cmr, Neos, Cms, Tcs, Rifs NifTAL*

TL16 (mutant) Nxr, dct, rpoN::Tn5, Neor This study

Plasmids

pSUP5011 Apr, Cmr, Neor, pBR322 derivative containing

Tn5-mob

Simon (1984)

pRK2013 Tra+, Kmr, ColE1 replicon Figurski and Helinski (1979)

pLAFR1 Cosmid cloning vector, Tcr Friedman et al. (1982)

pBlueScriptKS+ Apr, LacZ’, T7 Phil10 promoter, f1 ori Stratagene

pGEM-TEasy Apr, cloning vector Promega

pUS1 10Æ9-kb EcoRI fragment of rpoN::Tn5 insert

of strain TL16 in pBlueScriptKS+

This study

pUS2 1Æ527-kb PCR amplified fragment containing

rpoN gene of strain TAL620 in pGEM-TEasy vector

This study

pUS3 2Æ1-kb PCR amplified fragment containing rpoN

gene of strain TAL620 in a cosmid cloning vector pLAFR1

This study

*NifTAL, NifTAL project and MIRCEN, University of Hawaii, Paia, Hawaii, USA.

s, sensitive; r, resistant; Nx, nalidixic acid; Neo, neomycin; Km, kanamycin; Ap, ampicillin; Rif, rifampicin; Cm, chloramphenicol; Tp, trimethoprim;

Tc, tetracycline; Sm, streptomycine; dct, dicarboxylic acid transport defective.

U.S. Gautam et al. Mesorhizobium ciceri rpoN mutant

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1798–1807 1799

CaCl2.2H2O, 0Æ01, and sodium succinate, 5Æ0 or mannitol,

10Æ0 as carbon source, pH 6Æ8. Escherichia coli was grown in

Luria broth (Miller 1972). Antibiotic concentrations for

E. coli strain S17Æ1 ⁄ pSUP5011::Tn5-mob were as follows:

streptomycin, 50 lg ml)1; neomycin, 50 lg ml)1; and either

chloramphenicol, 30 lg ml)1 or ampicillin, 50 lg ml)1. For

growing M. ciceri, nalidixic acid was added at 25 lg ml)1

to the medium. The media were solidified with agar-agar

powder (Difco) at 15Æ0 g l)1 when necessary.

For enzyme assays, M. ciceri strains were grown in YM

medium for 2 days, washed and resuspended in 200 ml of

rhizobium minimal medium containing 10-mmol l)1 suc-

cinate and incubated for 6 h at 28�C on a shaker (Adolf

Kuhner AG, ISF-1-V, Schweiz) at 200 rev min)1 for the

induction of Dct activity.

Tn5 mutagenesis

For mutagenesis, the mating of the recipient strain

M. ciceri TAL620, grown in YM medium for 2 days at

28�C and the donor E. coli strain S17Æ1, harbouring the

suicide plasmid pSUP5011:: Tn5-mob (Simon et al. 1983;

Simon 1984), grown in Luria broth for 12 h at 37�C, was

carried out (Das et al. 2006). Transconjugants were selec-

ted on rhizobium minimal medium containing glucose

and succinate to which 2,3,5-triphenyl tetrazolium

chloride (30 lg ml)1), nalidixic acid (25 lg ml)1) and

neomycin (100 lg ml)1) were added. After 6 days of

incubation at 28�C, mutants defective in the utilization of

dicarboxylic acids were isolated by screening the nalidixic

acid–neomycin-resistant transconjugants, which appeared

as white colonies on succinate–glucose–tetrazolium plates

(Ronson et al. 1981), but failed to grow on succinate.

Growth

Wild-type M. ciceri TAL620 and the rpoN mutant strain

TL16 were analysed for their growth in rhizobium min-

imal medium containing mannitol (10 g l)1) as carbon

source. The cells were initially grown in YM medium,

harvested, washed with minimal medium and were used

as inocula. The growth of cells was monitored by measur-

ing the optical density at 600 nm in a SPECORD210

spectrophotometer, Analytik Jena, Germany.

Dicarboxylate uptake assay

For the induction of Dct activity, the cells were incubated

for 4 h in rhizobium minimal medium containing

10 mmol l)1 of either succinate or malate as appropriate.

Dicarboxylate uptake assays with induced M. ciceri

cells (300 lg of cell protein) were carried out at 30�C in

2-ml minimal medium (Bolton et al. 1986) containing

40 lmol l)1 [1,4-14C] succinate (specific activity 130 mci

;mmol)1) or 60 l mol l)1 [U-14C] malate (specific activity

192 mci mmol)1) in the presence of 5-mmol l)1 unlabelled

succinate or malate as appropriate. During incubation, an

aliquot of the sample was withdrawn at regular time inter-

val, passed through a filter (pore size, 0Æ45 lm; Millipore

Corp., Billerica, MA, USA), and washed twice each time

with 10 ml of cold minimal medium. The filters containing

the cells were air-dried and placed into vials containing

10 ml of scintillation fluid. 14C incorporation was counted

in a Beckman LS 8000 scintillation counter.

Preparation of cell-free extract

Cells grown in minimal medium were washed and suspen-

ded in 50-mmol l)1 tris-HCl buffer containing 1 mmol l)1

MgCl2, pH 7Æ4, for extraction of malate dehydrogenase or

50mmol l)1 potassium phosphate buffer (pH 7Æ4) for the

extraction of fumarase or succinate dehydrogenase. The

cells were disrupted by sonication with a Labsonic M

sonicator (B. Braun Biotech International, Melsungen,

Germany). The extract was centrifuged at 10 000 g for

30 min at 4�C and the clear supernatant was used as the

source of enzymes.

Enzyme assays

Enzyme assays were done in a spectrophotometer at 25�C.

Malate dehydrogenase was assayed following the method

of Yoshida (1969). Fumarase was assayed according to

Hill and Bradshaw (1969). Succinate dehydrogenase was

assayed following the protocol of Veeger et al. (1969). Dur-

ing enzyme assays, appropriate corrections were made for

substrate and enzyme blanks. Specific activities were

expressed as nanomoles of product formed per minute per

milligram protein. Protein was estimated using bovine

serum albumin as the standard (Bradford 1976).

DNA manipulation, cloning and sequencing

DNA was isolated according to standard protocol (Meade

et al. 1982). Routine manipulation of DNA, plasmid

isolation, construction of recombinant plasmids, electro-

phoresis of DNA and transformation were carried out

according to standard procedures (Sambrook et al. 1989).

Digestions with restriction enzymes and DNA ligation

were performed according to the manufacturers’ instruc-

tions (Promega, Inc. and New England Biochemical,

Beverly, MA, USA). Southern blotting was performed by

using a standard Southern blot protocol (Southern 1975)

as described previously (Das and Mishra 1996).

DNA sequencing was performed as described (Das

et al. 2006). Open reading frames (ORF) were identified

Mesorhizobium ciceri rpoN mutant U.S. Gautam et al.


ª 2007 The Authors

using BLASTX (Altschul et al. 1990). Comparisons of

DNA sequences and their derivative amino acid sequences

with sequences in the European Molecular Biology

Laboratory (EMBL) ⁄ GenBank were performed using

BLAST (Altschul et al. 1997) and CLUSTALX (Thompson

et al. 1994).

PCR amplification and cloning of the rpoN gene of

Mesorhizobium ciceri for complementation

The complete coding region of the rpoN gene from

M. ciceri strain TAL620 was amplified using the primers F1

(5¢-GAA GTC CGG ATG CGT AGA CGA GGC 3¢) and

R1 (5¢ TGG CTT GTG GAA AAT CGA CCC GCC-3¢),

and a 1527-bp product was obtained. The primers were

designed from the sequences of the flanking regions of

the rpoN genes of other bacteria in the database. PCR was

conducted, and the resulting DNA fragment was purified

using a QIAquick PCR purification kit (Qiagen, Hilden,

Germany) and cloned into pGEM-TEasy plasmid vector

(Promega) for sequencing as described (Das et al. 2006).

The oligonucleotide primers rpoNF (5¢- GCG GAA

TTC TGA AAT TTG TTT CCT GAC -3¢) and rpoNR (5¢-CCG GAA TTC AGG CCA ACG TCG TGA CGG -3¢)were used for PCR amplification of about 2Æ1 kb of

genomic DNA containing the rpoN gene (ORF2), intro-

ducing EcoRI site (underlined) into the PCR product.

After digestion with EcoRI, the PCR product was cloned

into the cosmid cloning vector pLAFR1 to yield pUS3.

Cosmid pSU3 was used to complement the mutant TL16.

Triparental spot mating was performed using the recipi-

ent TL16, the donor pSU3 and the helper strain

HB101 ⁄ pRK2013. The exconjugants were identified by

streaking on rhizobium minimal medium supplemented

with succinate or malate.

Genomic context analysis

Genomic context of rpoN gene of M. ciceri strain

TAL620 in comparison with the genomes of related

nitrogen-fixing members of rhizobiales were analysed

using the GeConT programme. This programme is avail-

able at: http://www.ibt.unam.mx/biocomputo/gecont.html

(Ciria et al. 2004).

Plant tests

Plant tests were carried out using surface-sterilized seeds

of chickpea (C. arietinum L) cv. Pusa C-235, as described

previously (Raychaudhuri et al. 2005). Each test was con-

ducted with three plants, and four replicates were consid-

ered for each test. At 35 days of inoculation, the shoot

portions of the plants were cut at the stem–root junction

and placed in an oven at 70�C for 5 days for dry weight

determination. Acetylene reduction assays were performed

(Ronson and Primrose 1979) using the nodules as des-

cribed previously (Das et al. 2006).

Microscopy

Root nodules were excised from the plants at 35 days of

inoculation and washed with 0Æ05-mol l)1 potassium phos-

phate buffer, pH 6Æ8. Slices from the central area of root

nodules were fixed in 2Æ5% glutaraldehyde in 0Æ05 mol l)1

potassium phosphate buffer, pH 6Æ8. Subsequent steps of

tissue preparations were carried out according to standard

protocol (Karnovsky 1965; Finan et al. 1983). Sections for

light microscopy were stained with 0Æ01% toluidine blue.

Ultra-thin sections for electron microscopy were stained

with 1% uranyl acetate, pH 4Æ5, followed by lead citrate

(Reynolds 1963) and examined with a Philips CM10

electron microscope.

Nucleotide sequence accession number

The nucleotide sequence data of M. ciceri reported in this

communication have been submitted to the GenBank

nucleotide sequence database under accession nos: ORF1,

EF079828; ORF2, AY850034; ORF3, EF460345; ORF4,

EF460347; and ORF5, EF460346.

Results

Transposon mutagenesis and isolation of mutant

Tn5 was introduced into M. ciceri strain TAL620 using the

suicide plasmid pSUP5011 containing Tn5-Mob from

E. coli S17Æ1 developed by Simon et al. (1983). Transconju-

gants occurred at a frequency of 4Æ2 · 10)4 per donor.

Spontaneous neomycin (100 lg ml)1)-resistant colonies of

M. ciceri strain TAL620 occurred at a very low frequency

(<10)9). The selection for the M. ciceri transconjugants was

carried out on YM-agar plates containing neomycin and

nalidixic acid. Over 5000 nalidixic acid–neomycin-resistant

transconjugants were obtained. Screening on minimal

medium containing glucose and succinate as carbon

sources and 2,3,5-triphenyl tetrazolium chloride allowed

the isolation of several white colonies of mutants, of which,

TL16 (Table 1) was selected for further studies.

Phenotypes of Mesorhizobium ciceri mutant

A phenotype of the M. ciceri mutant TL16 was its failure

to grow with succinate, malate or fumarate as carbon

source; whereas growth with mannitol was similar to that

of the wild type (Fig. 1). Similar results were obtained


ª 2007 The Authors


with pyruvate also. The mutant TL16 grew at approxi-

mately wild type rates with glucose, galactose, sucrose,

ribose, citrate, arabinose or ribose as carbon source.

The mutant was examined for its ability to take up

succinate as compared with the wild type. While the wild

type strain took up succinate efficiently, the mutant

showed negligible uptake activity (Fig. 2a). Similar results

were obtained for malate also (Fig. 2b).

Extracts of M. ciceri wild-type strain TAL620 and of

the mutant TL16 were assayed for activities of succinate

dehydrogenase, malate dehydrogenase and fumarase

which use C4-dicarboxylate as substrate to examine if

mutation in the rpoN gene had any bearing on the activ-

ity of these enzymes. While the wild-type parent had con-

siderable activities, the mutant had practically no activity

of these enzymes (Table 2).

Cloning and physical localization of the gene

complementing TL16

The EcoRI fragment containing the transposon (Fig. 3a)

was cloned into the pBlueScript vector, transformed into

E. coli strain DH5a and the recombinant plasmid pUS1

was isolated. The nucleotide sequence of the DNA flank-

ing the transposon in the plasmid pUS1 was determined

by dideoxy chain termination method. The sequence

revealed three complete and two partial ORF. Database

sequence similarity searches showed that, in the mutant

Tn5 interrupted a gene homologous to those coding for

RpoN, which is an alternative sigma factor (r54) of differ-

ent organisms. The gene was flanked by conserved ORF,

one (ORF1) upstream and three (ORF3, ORF4 and

ORF5) downstream (Fig. 3b). All the ORF, except ORF5,

are transcribed in the same direction. The ORF1 localized

just upstream of rpoN, encodes a product with similarity

to ATP-binding subunits of ABC transporters. The ORF2,

which is the M. ciceri rpoN sequence extends over

1527 bp and encodes a protein of 508 amino acids.

A comparison of the deduced amino acid sequence of the

rpoN gene with those in the databases revealed a high

degree of similarity with the RpoN from different sources.

The N-terminal region of the deduced protein product

has the domain of 50 amino acids, rich in leucine. The

carboxy terminal region exhibits the helix-turn-helix

structure (amino acid positions 400–420, M. ciceri count)

and the invariant sequence ARRTVAKYRE (position 477–

486), known as the RpoN box. The deduced protein

product shares 89%, 58%, 57%, 56%, 55%, 52% and 49%

identities with those of Mesorhizhobium loti, Mesorhizobi-

um sp. BNCI, S. meliloti, Rhizobium sp. strain NGR234,

Rhizobium leguminoserum bv. viciae, Azorhizobium cauli-

nodans, R. etli, B. japonicum, respectively. The ORF3

located immediately downstream of rpoN shows homol-

ogy with the gene for r54 modulation protein, the ORF4

10

Time (min)

30(a)

25

20

15

10

5

00 108642

[14C

] Suc

cina

te u

ptak

e[n

mol

(m

g pr

otei

n)–1

]

(b)50

40

30

20

10

00 8642

Time (min)

[14C

] Mal

ate

upta

ke[n

mol

(m

g pr

otei

n)–1

]

Figure 2 Dicarboxylic acid uptake by wild-type Mesorhizobium ciceri

strain TAL620 and the mutant strain TL16. (a) [14C] Succinate uptake;

(b) [14C] malate uptake. (r), M. ciceri TAL620; ( ), TL16.

Time (h)

O.D

. at 6

00 n

m

0·5

0·4

0·3

0·2

0·1

00 20 40 60 80

Figure 1 Effect of mannitol on the growth curves of the Mesorhiz-

obium ciceri wild-type strain TAL620 (s) and the mutant strain

TL16 (d).

Table 2 Activity of the enzymes using dicarboxylates as substrate in

the wild-type Mesorhizobium ciceri strain TAL620 and the mutant

TL16

Strains

Specific activity (nmol min)1 mg)1 of protein)

Succinate

dehydrogenase

Malate

dehydrogenase Fumarase

TAL620 (wild type) 82Æ0 193Æ0 46Æ0

TL16 1Æ0 0Æ5 0Æ0



ª 2007 The Authors

with the gene of phosphotransferase system enzyme II

and the ORF5 with that of NADH oxidase.

A 1Æ527-kb stretch of chromosomal DNA containing the

ORF2 (rpoN gene) was amplified from the wild-type strain

TAL620 by PCR. It was cloned into pGEM-TEasy vector;

subsequently, the recombinant plasmid pUS2 was isolated

and the rpoN gene in the clone was sequenced. The location

of the Tn5 mutation was determined from the sequence of

the transposon insertion junction in the rpoN gene frag-

ment, which was earlier amplified by PCR from the DNA

of the wild-type strain, TAL620 and sequenced. It was

found that Tn5 insertion was within the codons 150–153.

The genomic context of rpoN gene in M. ciceri, as com-

pared with that in the related nitrogen-fixing members of

Rhizobiales shows that it is almost identical to that of

M. loti, although considerable homogeneity in composi-

tion was also observed with those of B. japonicum, M. loti

and R. etli. All the four genomes are observed to have

gene orthologs for ATP-binding protein, sigma 54 pro-

tein, sigma 54 modulation protein and phosphotransf-

erase system enzyme II in their genome (Fig. 3c).

For confirmation, complementation of mutant TL16

with the ORF coding for rpoN gene was carried out.

A 2Æ1 kb DNA fragment containing rpoN gene was ampli-

fied by PCR from the strain TAL620 and cloned into

pLAFR1 to yield pUS3. The cosmid pSU3 was used to

complement the mutant TL16 by triparental mating to

yield the exconjugant TL16 ⁄ US3 (Table 3). The cosmid

pUS3 was able to complement TL16, and exhibited pheno-

type similar to that of the wild type in respect to growth

B. japonicum

Tn5-mob (7·7 kb)

EcoRI

EcoRI

Allur1

Pst1

Sma1

Bgl1Sal1 Sac2

Pvu1

Sal1

Msc1

EcoRI

EcoRI

2·0 kb

(a)

(b)

(c)

10·9 kb

3·2 kb3210

ORF3ORF1

blr0722

RHE

ABC

rpoN2 bl

ml

RHE

smp

m

p

NADH

NADH

RH

p b

mll3196

rpoNch

rpoN ptsll

mll3

ORF2 (rpoN) ORF4 ORF5

M. loti

R. etli

M. ciceri

Figure 3 Genetic organization and genomic context of the Mesorhizobium ciceri mutant strain TL16 rpoN DNA region. Panel a, the EcoRI-diges-

ted DNA fragment containing the transposon Tn5; panel b, the restriction sites and transcriptional directions of ORF1, ORF2 (rpoN), ORF3, ORF4

and ORF5; panel c, genomic context and organization conservation of the rpoN region in the genome of M. ciceri in comparison with genomes

of related nitrogen-fixing members of Rhizobiales. Arrows represent genes with their relative orientation in the genomes. Genes are coloured

according to their functional categories and labelled according to their original gene annotation in the database. Yellow, ATP-binding protein;

orange, sigma 54 protein; red, sigma 54 modulation protein; green, phosphotransferase system enzyme II; grey, putative permease protein; light

brown, NADH oxidase; and pink, acetyl transferase.

Table 3 Symbiotic properties of the wild-type Mesorhizobium ciceri

TAL620 and the rpoN mutant TL16

Strains

Shoot dry

weight (g)*

Acetylene

reduction�

TAL620 1Æ402 ± 0Æ27 1959 ± 128Æ4

TL16 0Æ517 ± 0Æ11 –

TL16 ⁄ US3 (TL16 containing pUS3) 1Æ29 ± 0Æ18 1928 ± 119

Uninoculated control 0Æ496 ± 0Æ13 –

*Dry weight is expressed as the mean values in g ± the standard devi-

ation of four replicates, each with three plants.

�Acetylene reduction activities are expressed as the mean of nano-

moles of ethylene formed per hour ± the standard deviation of four

replicates, each with three nodulated plant roots.

–, not detectable.


ª 2007 The Authors


on succinate or malate and the acetylene reduction

activity (Table 3).

When genomic DNA from the strain TL16 was digested

with EcoRI restriction enzyme and hybridized with 32P-

labelled internal HindIII fragment (�5Æ4 kb) of Tn5-mob

as probe, a positive signal was obtained on a 10Æ9 kb

DNA fragment of TL16 (Fig. 4a). To examine the copy

number of rpoN gene present in M. ciceri, genomic DNA

of the strain TAL620 or TL16 was digested with EcoRI

and hybridized with the 1Æ527 kb PCR amplified rpoN

gene sequence as probe. Only one hybridization band

signal was observed either on a 10Æ9 kb EcoRI DNA

fragment of TL16 or on a 3Æ2-kb EcoRI DNA fragment

of TAL620 (Fig. 4b).

Symbiotic properties of the rpoN mutant

The symbiotic properties of the mutant TL16 was investi-

gated after inoculation of chickpea seedlings growing

under nitrogen-deficient condition. Plants inoculated with

the strain TL16 were pale green. At 35 days, the mean dry

weight of the shoots of the plants (1Æ402 g per three

plants) inoculated with the wild-type strain TAL620 was

almost three times that of the plants (0Æ517 g per three

plants) inoculated with the mutant strain TL16. The

mean dry weight of the plants inoculated with the mutant

was, however, not appreciably different from that of the

uninoculated control (Table 3). The nodules formed by

the wild-type strain had a high acetylene reductase activ-

ity (Table 3). In contrast, the acetylene reduction activity

of the nodules formed by the rpoN mutant was absent

(Table 3).

Under nitrogen-deficient condition, nodules induced

by the wild-type strain TAL620 were pink and spherical

(Fig. 5a). In contrast, nodules induced by the mutant

TL16 were small, white and often wrinkled (Fig. 5b).

The control plants without bacterial inoculation, as

expected, had no nodules on their roots (Fig. 5c). Nod-

ules formed by the rpoN mutant M. ciceri TL16 were

examined to determine if the absence of nitrogen fix-

ation was accompanied by any abnormality in nodule

structure. Light microscopic observations revealed that

the nodules contained a large number of empty, un-

infected cells, while only a few infected host cells were

present within a poorly developed symbiotic zone. The

infected cells along with the neighbouring uninfected

cells contained many starch granules near their peripher-

ies as compared with the nodules induced by the wild

type (Fig. 5d,e). Electron microscopic observations

revealed that the nodules induced by the wild type con-

tained mature bacteroids surrounded by a PBM and also

poly-hydroxybutyrate granules. In these nodules, the host

cytoplasm was homogeneous and very electron dense. In

addition, the bacteroids had proliferated to the extent

that most of the host cytoplasm was occupied by them

(Fig. 5f). However, infected cells in nodules induced by

the strain TL16 appeared to degenerate shortly after the

bacteroids were formed. The infection tubes were seen

containing lysed bacteria. Most of the bacteria failed to

transform into bacteroids. Moreover, amyloplasts, mito-

chondria, endoplasmic reticulum and the remnants of

other organelles and membranes were found in the host

cell cytoplasm (Fig. 5 g–i).

Discussion

Cicer arietinum is the third most widely grown grain

legume in the world, and is grown extensively in the

Middle East and many regions of India (van der Maesen

1972). It is a tropical legume and is nodulated by

M. ciceri. To study the genetic basis of Dct in M. ciceri

and its implications in symbiotic performance of the

bacteria, the mutants were raised by Tn5 mutagenesis of

the wild-type strain TAL620. Putative dicarboxylate

mutants were then selected as white colonies on plates

containing glucose and succinate as carbon sources

and 2,3,5-triphenyl tetrazolium chloride. Dicarboxylate

uptake studies with one of the mutants, TL16, con-

firmed the inability of the mutant to transport dicarb-

oxylates, indicating the absence of a functional Dct in

the strain. Although the mutant fails to grow on dicarb-

oxylates, its growth similar to that of the wild type on

other carbon sources, such as pyruvate, glucose, galac-

tose, sucrose, ribose, mannitol, citrate, arabinose or

ribose, indicates that it has a functional tricarboxylic

acid cycle (Bolton et al. 1986). The absence of the activ-

ity of the enzymes that use C4-dicarboxylate as substrate

M (b)kb

10·9

3·2

23·0

4·3

9·4

6·5

M(a)

kb

321 21

Figure 4 Southern hybridization analysis. (a) localization of the site

of Tn5 insertion in Mesorhizobium ciceri mutant strain TL16. Lane 1,

strain TAL620; lane 2, TL16. (b), rpoN containing DNA sequence in

TAL620 (wild type). Lane 1, TL16; lane 2, strain TAL620; and lane 3,

plasmid pUS1.



ª 2007 The Authors

in the extract of the mutant indicates inducible nature

of these enzymes in M. ciceri.

Cloning and sequencing of the DNA fragment con-

taining the transposon revealed that Tn5 had inter-

rupted the gene for RpoN which is an alternative

sigma factor. A single band as observed following

Southern hybridization of the wild-type DNA with rpoN

probe suggests the presence of only a single copy of

rpoN in M. ciceri TAL620. This is in contrast to M. loti

strain MAFF303099, R. etli and B. japonicum, where

two copies of rpoN gene (rpoN1 and rpoN2) were

observed (Kullik et al. 1991; Michiels et al. 1998a;

Kaneko et al. 2000; Dombrecht et al. 2002) and also

A. caulinodans, where the presence of two copies of

rpoN were conjectured (Stigter et al. 1993). Thus, the

mutant phenotype of M. ciceri TL16 is attributed to

the inactivation of the rpoN gene by Tn5 insertion on

a single site in the chromosomal DNA. Genomic con-

text study reveals that the rpoN gene in M. ciceri

has an organizational conservation identical to that of

M. loti and largely similar to that of B. japonicum and

R. etli.

The white nodules incited by the mutant strain

M. ciceri TL16 indicated that these were ineffective, and

indeed, the pale green colour of the hosts pointed to a

lack of significant nitrogen fixation by the bacteroids in

their nodules. This was further confirmed by the failure

of acetylene reduction by the nodules induced by the

mutant M. ciceri TL16, which also resulted in reduced

dry weight of shoots and not significantly different

from that of the uninoculated control plants. Thus, it

appears that rpoN function is required for symbiotic

nitrogen fixation in M. ciceri. The presence of a large

number of empty, uninfected cells in the symbiotic

0·5 µm

b

pbmvs

ermt

am

sb

lbit

0·1

0·1 µm

phb

sbb

mt

0·6 µm

0·3 µm

0·3 µm

b

ncs

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 5 Morphology of nodules formed on chickpea roots by wild-type Mesorhizobium ciceri (TAL620) and rpoN mutant TL16. Nodules shown

were photographed at 35 days of inoculation with: (a) Mesorhizobium ciceri TAL620; (b) rpoN- mutant TL16; (c) uninoculated control. Light micro-

graphs of sections of the symbiotic zones of nodules elicited by: (d) TAL620 (wild type); (e) mutant TL16. b, bacteroids; s, starch granule; nc, nod-

ule cortex. Electron microscopic sections of nodules elicited by: (f) TAL620 (wild type); (g), (h) and (i) mutant TL16. vs, gas exchange vesicle; b,

bacteroids; mt, mitochondria; am, amyloplast; er, endoplasmic reticulum; pbm, peribacteroid membrane; sb, senescent bacteroid; phb, granules

of polyhydroxybutyrate; it, infection tube; lb, lysed bacteria.


ª 2007 The Authors


zone of nodules formed by the mutant M. ciceri TL16

and the failure of infecting bacteria to transform into

bacteroids indicate the role of rpoN function in the

maturation of bacteroids and subsequent nitrogen

fixation by them.

Acknowledgements

We are grateful to Prof F.M. Ausubel (Department of

Genetics, Harvard Medical School, Boston, Massachusetts,

USA) for providing the cosmid pLAFR1. We thank

Prof D.H. Figurski (Department of Biology, University of

California, San Diego, La Jolla, USA) for providing the

strain pRK2013. We are grateful to All India Institute of

Medical Sciences, New Delhi, for Electron Microscopic

Facility. We are thankful to the Distributed Information

Sub Center at Institute of Life Sciences, Bhubaneswar,

for making the genetic map of the rpoN gene sequences.

We are grateful to the Dept. of Biotechnology, Govt. of

India, for financial assistance.

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