Fec1 and Sigma Factor

Regulation of the FecI-type ECF sigma factor bytransmembrane signallingVolkmar Braun�, Susanne Mahren and Monica Ogierman

Induction of the ferric citrate transport genes of Escherichia coli

K-12 involves a signalling cascade that starts at the cell surface

and proceeds to the cytoplasm. Three specific proteins are

involved: FecA in the outer membrane, FecR in the cytoplasmic

membrane, and FecI in the cytoplasm. The binding of dinuclear

ferric citrate to FecA causes substantial structural changes in

FecA, triggering the signal cascade. The amino-proximal end of

FecA interacts with the carboxy-proximal end of FecR in the

periplasm. FecR then transmits the signal across the

cytoplasmic membrane into the cytoplasm and activates the

FecI sigma factor, which binds to the RNA polymerase core

enzyme and directs the RNA polymerase to the promoter

upstream of the fecABCDE transport genes to initiate

transcription. Transcription of the fecIR regulatory genes and the

fec transport genes is repressed by the Fur protein loaded with

Fe2þ. Therefore, transcription of the fec transport genes is

subjected to double control: cells first detect iron deficiency and

respond by synthesis of the regulatory proteins FecI and FecR,

which initiate transcription of the fec transport genes, provided

ferric citrate is available. FecI belongs to the extracytoplasmic

function sigma factors, which are widespread among bacteria.

With the recent sequencing of complete microbial genomes, it

has become apparent that the FecIRA cascade is now a

paradigm for the regulatory control of FecI family sigmas in

Gram-negative bacteria.

AddressesMikrobiologie/Membranphysiologie, Universtat Tuebingen, Auf der

Morgenstelle 28, 72076 Tuebingen, Germany�Correspondence: V Braun;

e-mail: [email protected]

Current Opinion in Microbiology 2003, 6:173–180

This review comes from a themed issue on

Cell regulation

Edited by Andree Lazdunksi and Carol Gross

1369-5274/03/$ – see front matter

� 2003 Elsevier Science Ltd. All rights reserved.

DOI 10.1016/S1369-5274(03)00022-5

AbbreviationsECF extracytoplasmic function

Fec ferric citrate

IntroductionIn 1994, Lonetto et al. [1] recognised that a group of

transcription regulatory proteins were in fact sigma (s)

factors which regulate genes that determine cell envelope

functions and for this reason were designated extracyto-

plasmic function (ECF) s factors; Fec I was among this

group. With genome sequencing has come the realisation

that not all of the ECF sigma factors regulate extracyto-

plasmic functions. Hence, Helmann has proposed renam-

ing these as the ‘group-4 sigmas’. The group-4 sigma FecI

regulates the ferric citrate (Fec) transport system of

Escherichia coli K-12 In turn, FecI is regulated by FecA

and FecR. FecA is an outer membrane protein that

receives the signal at the cell surface and transmits the

signal across the outer membrane into the periplasm. The

amino-terminal extension of FecA, which is not found in

other active outer membrane transport proteins of E. coliK-12, is essential for signalling to FecR. FecR is a

cytoplasmic membrane protein that transmits the signal

across the cytoplasmic membrane and regulates the activ-

ity of FecI. The complete signalling cascade including

the signal, the signal receptor, transfer of the signal across

the outer membrane and the cytoplasmic membrane, and

the receiver in the cytoplasm has only been fully eluci-

dated in the E. coli Fec transport gene regulation system.

The activity of outer membrane transport proteins

requires energy that is derived from the proton motive

force of the cytoplasmic membrane. The coupling device

between the outer membrane and the cytoplasmic mem-

brane consists of a protein complex composed of TonB,

ExbB and ExbD. TonB contacts the transport proteins

and converts them into active transporters. FecA requires

TonB not only for transport of ferric citrate across the

outer membrane but also for transmitting the regulatory

signal across the outer membrane, contacting TonB

through its TonB box, a heptapeptide at the carboxy-

terminal border of its amino-terminal external extension.

A BLAST search reveals that FecI, together with other sfactors, form a subgroup of ECF s factors with a score of

more than 123 (see the lower branch in Figure 1). Only sfactors of Gram-negative bacteria — Pseudomonas spe-

cies, Bordetella species and Xanthomonas campestris, all of

which are potential pathogens for humans, animals and

plants — belong to the group. The upper branch of

Figure 1 includes mainly Pseudomonas species but also

Agrobacterium tumefaciens, Bordetella bronchiseptica and

Caulobacter crescentus. ECF s factors of streptomycetes

and mycobacteria (which are not included in Figure 1 and

will not be discussed here) are also in the BLAST score

window (51–91).

In this review, we discuss signal transduction from the cell

surface to the cytoplasm which ultimately results in an

active FecI ECF that initiates transcription of the ferric

173

www.current-opinion.com Current Opinion in Microbiology 2003, 6:173–180

citrate transport genes of E. coli K-12. This system has been

characterised in the most detail and exhibits properties that

will be typical for the whole class of FecI-type ECFs.

Evidence for an extracytoplasmic regulationmechanismAdding 1 mM of ferric citrate to growth medium induces

the formation of the ferric citrate transport system [2].

Both the fact that citrate accumulated intracellularly (in

an icd mutant devoid of isocitrate dehydrogenase activity)

does not induce the Fec transport system [2] and that

transport studies reveal it is iron, and not citrate, that is

transported into the cytoplasm of cells clearly show that,

for induction, ferric citrate is not taken up into the

cytoplasm but acts from outside the cell.

The FecI systemFecI regulates transcription of the fecABCDE transport

genes for ferric citrate in E. coli. The fecIR regulatory

genes are located upstream of the fecABCDE genes and

form a separate transcriptional unit (Figure 2) [2]. fecAencodes an outer-membrane transport protein and

fecBCDE an ABC transport complex of the cytoplasmic

membrane (Figure 2) [3]. Both fecIR and fecABCDE

Figure 1

PA4896P. aeruginosa

FecIE. coli

FecIX. campestris

PA2468 P. aeruginosa

PA1912 P. aeruginosa

RhuI B. avium

PupIP. putida

PA0472P. aeruginosa

FiuIP. aeruginosa

PrhIR. solanacearum

PA2050P. aeruginosa

PA2093P. aeruginosa

PfrIP. aeruginosa

PvdSP. aeruginosa

PbrAP. fluorescens

PsbS P. B10

PvdS P. fluorescens

ATU5309A. tumefaciens

PA2896P. aeruginosa PA3410

P. aeruginosa

CC2707C. crescentus

PAO149P. aeruginosa

PA1300P. aeruginosa

PA2387P. aeruginosa

ATU3692A. tumefaciens

BupIB. bronchisepticum

CC0981C. crescentus

HurI B. pertussis

PA3899P. aeruginosa

Current Opinion in Microbiology

Phylogenetic tree demonstrating the degree of sequence identities among ECF sigma factors related to the E. coli K-12 fecI gene, as revealed by

analysis with Clustal W.

174 Cell regulation

Current Opinion in Microbiology 2003, 6:173–180 www.current-opinion.com

Figure 2

Cytoplasmicmembrane

Fe+2ADP+PiADP+Pi

ATPATP

fecABCDEPFur PfecAfecIR

160160

C2

C1

Outermembrane

FecA unloaded FecA loaded

L8L7

TonBbox

TonBfragment

FecI

RNAPcoreenzyme

FecB

N N N

N

N

C

C

N

(Fe3+citrate)2

PFur PecI

Fur

FurFe2+FurFe2+

Fe2+

Exb

D

TonB

2

N

Exb

B

TonB

1σ2

σ3

σ4

Fec

R

C

LLLV

Fec

D

Fec

C

FecE FecE


Model of the ferric citrate transport and regulatory systems. The large structural changes in loops 7 (L7) and 8 (L8) that occur upon binding of dinuclear

ferric citrate are shown. The movement of these loops closes off the cavity containing dinuclear ferric citrate from the external environment. The

unknown conformation of the amino-proximal region of FecA, marked N, is shown to interact with region 160 of TonB. The figure shows the crystal

structure of the TonB fragment. This is a dimer, although evidence exists to show that complete TonB forms a monomer [21]. The transmembranetopology of the energy transducing the TonB–ExbB–ExbD protein complex is shown, but the model does not reflect the unknown stoichiometry of the

complex. A 7:2:1 ratio of ExbB : ExbD : TonB has been determined in cells but not in an isolated complex [22]. Interactions between FecA and the

heptad leucine-zipper-like motif (marked LLLV) of FecR, between the amino-terminal region of FecR and region 4 of FecI [23], and between FecI and

the RNA polymerase (RNAP) core enzyme (S Mahren and V Braun, unpublished data) are shown by arrows. The FecI–RNAP (RNA polymerase)

holoenzyme binds to the promoter of fecA. The Fur protein loaded with Fe2þ (Fur Fe2þ) binds to the Fur boxes (PFur) upstream of fecI and fecA and

represses fecIR and fecABCDE transcription. The ferric citrate transport system (ABC transporter) is shown on the left-hand side of the figure.

Regulation of the FecI-type ECF sigma factor by transmembrane signalling Braun, Mahren and Ogierman 175


transcription is repressed by the Fur protein loaded with

ferrous iron. Under iron-limiting growth conditions result-

ing in low intracellular iron concentrations, Fur is

unloaded and transcription of fecIR occurs. By contrast,

iron limitation is not sufficient to induce transcription of

the fecABCDE transport genes. Rather, both iron limita-

tion and the presence of ferric citrate are required for

fecABCDE transcription. FecA transports iron citrate and

interacts with FecR to allow FecI activation (see below).

High expression of fecA guarantees that a minimal level of

FecA is present in uninduced cells to respond to ferric

citrate and initiate the regulatory cascade. When an

intracellular iron surplus is reached, ferrous iron binds

to Fur, which represses transcription of the fecIR and

fecABCDE genes [2].

FecI incubated with purified RNA polymerase is suffi-

cient to bind to the fecA promoter and initiate transcrip-

tion [2]. The exclusive use of FecI in fec transport gene

regulation was shown in a study in which the seven E. colis-factors, s70, sN, sH, sF, sE, sS and purified FecI, were

incubated with the RNA polymerase core enzyme. Only

FecI RNA polymerase transcribed a DNA fragment con-

taining the fecA promoter [4].

Interaction of regulatory proteins in thesignalling cascade from cell surface tocytoplasmTranscription regulation of the fec transport genes is

accomplished by three regulatory proteins: FecA, FecR

and FecI. FecA serves two functions: induction of fectransport gene transcription, and transport of ferric citrate.

Various mutants indicate that these two functions can be

uncoupled. Binding of ferric citrate to FecA without

transport is sufficient to initiate the signalling cascade

that finally leads to transcription of the fec transport genes.

The crystal structure of the FecA protein reveals that the

ferric citrate form that binds to FecA is dinuclear ferric

citrate (Fe3þ citrate)2 [5��], thus definitely identifying the

inducing species. (Fe3þ citrate)2 binds to ten residues of

FecA, which are located in a cavity that lies well above the

outer boundary of the outer-membrane lipid bilayer.

Binding of (Fe3þ citrate)2 induces strong long-range

structural transitions in FecA. Surface loops 7 and 8 are

translated 11 A and 15 A, respectively, and cover the entry

of the surface cavity, presumably preventing the escape of

(Fe3þ citrate)2 back to the external milieu. In the FecA

region exposed to the periplasm, a short helix is unwound.

Smaller translations of 2 A and less are observed in several

other regions of FecA. The massive structural changes in

FecA presumably generate a signal that is transmitted

across the outer membrane. However, the structural

changes that occur upon binding of (Fe3þ citrate)2 to

FecA are not sufficient to initiate the transcription initi-

ating signalling cascade. Signalling is dependent on TonB

activity, as is (Fe3þ citrate)2 transport across the outer

membrane. It is assumed that additional structural

changes occur through interactions with TonB, or that

the structural changes induced by (Fe3þ citrate)2 binding

are modified by TonB.

Because the signal initiated in FecA needs to reach the

cytoplasm where transcription of the fec genes takes place,

the signal must cross the periplasmic space and the

cytoplasmic membrane. Crossing the periplasm is

achieved by interaction of the amino-terminal end of

FecA with the carboxy-terminal end of FecR; both of

which are localised in the periplasm (Figure 2) [6,7]. The

appealing concept that the amino-terminal end of FecA

interacts in the periplasm with the carboxy-proximal end

of FecR was demonstrated by a bacterial two-hybrid

system that revealed specific interaction of FecA1–79

(residues 1–79 of mature FecA) with FecR101–317 [8].

FecR contains a motif composed of repeating heptapep-

tides (residues 247–268) that are flanked by three leucine

residues and one valine residue (Figure 3). It resembles

leucine zipper motifs. The motif is highly conserved in

FecI-like ECF anti-s factors (Figure 3). Replacement of

the leucine and valine residues by proline results in

derivatives with a strongly decreased interaction with

FecA and a low fecA transcription in response to ferric

citrate (S Enz and V Braun, unpublished data). Either the

conformation of the repeat sequence and/or the leucine

residues are important for interaction with FecA.

Analogous in vivo experiments using the LexA system

demonstrate binding of FecI to FecR1–85, FecR1–58 and

FecR9–85 [8]. Residues 9–58 seem to be sufficient for the

binding of FecR to FecI. In vitro, FecR–(His)6 (six

histidine residues fused to the carboxy-terminal end of

FecR) bound to a Ni-agarose column binds FecI [8].

Signal transfer across the outer membrane by FecA,

interactions between FecA and FecR in the periplasm,

signal transfer across the cytoplasmic membrane by FecR,

and interactions between FecR and FecI in the cytoplasm

form a complete signalling cascade from the cell surface to

the cytoplasmic site of transcription initiation.

Sequence analysis and biochemical studies reveal that sfactors, including ECF s factors, are divided into struc-

turally and functionally conserved subregions (Figure 4).

A FecI deletion analysis using the LexA two-hybrid

system reveals that regions 4.1 and 4.2 of FecI interact

with FecR1–85 [9]. Additional mutagenesis and overex-

pression studies support this conclusion.

Mechanism of ferric citrate transcriptionregulationFecI recruits the RNA polymerase core enzyme and

directs it to the promoter of the fecA gene, which is the

major or only promoter of the fecABCDE transport gene

transcription. The activity of most ECF s factors seems to

be controlled by anti-s factors. In the absence of anti-s

176 Cell regulation


factors, the s factors initiate transcription without extra-

cytoplasmic signals.

There is no evidence that FecR acts as an anti-s factor. In

the absence of FecR, there is virtually no fecABCDEtranscription. By contrast, cells containing the cytoplas-

mic FecR1–85 and even fragments of FecR1–85 display a

high constitutive transcription of the fecABCDE genes.

Cells containing longer FecR derivatives — which extend

from residue 1 to 273 (out of a total of 317 residues) [2] —

that do not interact with FecA [8] also transcribe

fecABCDE constitutively, but to a lower level. Although

FecR is necessary for FecI activity, it cannot be ruled out

that FecR acts as an anti-s factor. If FecI is unstable,

spontaneously denatures, precipitates or is degraded by

proteases, binding to FecR could maintain FecI in a

stable conformation. When the signal from FecA occu-

pied by (Fe3þ citrate)2 arrives through FecR, FecR

undergoes a conformational change that may result in

the dissociation of FecI from FecR and immediate bind-

ing of FecI to the RNA polymerase core enzyme. In this

model, FecR acts as both a chaperone for FecI and as an

anti-s factor, given that FecI is kept in an active con-

formation or assumes an active conformation with FecR

but cannot exert activity while it is bound to FecR.

Transcription regulation of the Fec type inspecies other than E. coliPseudomonas putida WCS358 expresses an iron transport

system via the siderophore pseudobactin BN8, which

Figure 3

TMCytoplasmic domain Periplasmic domain

LLLVN C FecR

247 254 261 268FecR E.coli WTKDILSFSDKPLGEVIATLTRYRNGVLRCDPFecR X. campestris WERGQLIADELRLDAFVAELERYRPGLLRCDPPupR P. putida WSQGMLVAQGQPLAAFIEDLARYRRGHLACDPFiuR P. aeruginosa WAQGMLVVENARLADLVAELGRYSPALLQVDPPA0471 P. aeruginosa WAQGMLVVENARLADLVAELGRYSPALLQVDPPA1911 P. aeruginosa WTRGMLMADRMPLAEVLAELARYRRGVLRCDPPA2051 P. aeruginosa WLDGRLEVRDRPLGEVLEALRAYRRGIISVADPA3409 P. aeruginosa WRRGLLVFDEQPLGEVVARLNRYRPGHLLVAPContig312 P. fluorescens WDKGMLLASNMRLDELLGELSRYRRGVLRCHPContig259 P. fluorescens WVQGRLEVRDRPLSEVIDSLRSYRRGILHLSPFrag10708 P. putida WTEGVLSVQQMPLAEFASELGRYRPGLLRCAPFrag10749 P. putida WEHGMLLARDMRLADLLQELARYRPGVLRCHPFrag10715 P. putida WREGALRLDDRPLGELLHELRRYRPGVLRWAPFrag3802 P. syringae WTRGVLKVDDQPLSEVLQTLATYRHGLLRYDTContig812 B. pertussis WEDGLLVVHGWRLDRLAAQLARYRLGVIRVDPContig1034 B. pertussis WEDGLLVVHGWRLDRLAAQLARYRLGVIRVDPRhuR B. avium WLRGVLHVNAMPLAAFAAELGRYRRGLVRCAQ * . * .*. * *. ...


1 84 101 246 269 317

Sequence comparison of predicted FecR-like proteins in the region LLLV through which FecR interacts with FecA (S Enz and V Braun, unpublished

data). The conserved leucine residues (L), which form the flanking residues of four heptad repeats, are shown in grey. The asterisks represent identical

amino acids in all listed proteins; full stops (.) represent identical proteins in most of the listed proteins.

Figure 4

1.2N C FecI

Binding site of FecR9-59

2.1 2.2 2.42.3 4.13.1 3.2 4.21.2

1 9 84 113 173

-10 recognition -35 recognition


Domain structure of the FecI s factor. Fecl lacks the 1.1 region of s70 factors. The regions through which Fecl binds to the �10 and �35 promoter

regions and to FecR9–59 (residues 9–59 of FecR) are shown.



induces synthesis of the PupB outer-membrane transpor-

ter [10]. Two genes, pupI and pupR, are located upstream

of pupB (Figure 5). Transcription induction of pupB by

pseudobactin BN8 requires pupI and pupR. pupI shows

42.8% and pupR 36.6% sequence identity to fecI and fecR,

respectively. Although regulation of pupB transcription

resembles regulation of fecABCDE transcription, PupR

might function only as an anti-s factor, given that PupI in

the absence of PupR induces pupB transcription consti-

tutively. In light of the data collected with the Fec

system, one may conclude that the structural change in

PupA upon binding of pseudobactin 358 is mediated

through the amino-terminal extension of PupB to PupR,

which then dissociates from PupI, releases PupI and acts

as a pupB-specific s factor.

In Pseudomonas aeruginosa, synthesis of the siderophore

pyoverdin and transport of ferric pyoverdin is controlled

by a FecIR-like regulatory device [11]. Pyoverdin (pre-

sumably after loading with Fe3þ) functions as an inducer

of the pyoverdin synthesis and Fe3þ pyoverdin transport

genes and induces formation of exotoxin A and an extra-

cellular endoproteinase. In this system, the FecR homo-

logue fpvR and the fpvA gene (equivalent to fecA) map

close to the pyoverdin synthesis operon, whereas the fecIhomologue pvdS lies some distance away (Figure 5). PvdS

is an ECF s factor and is required for the synthesis of

pyoverdin, exotoxin A and endoproteinase. Synthesis is

high in fpvR mutants; overexpressed fpvR inhibits synth-

esis, indicating that FpvR functions as an anti-PvdS sfactor. Furthermore, FpvA is required for the induction

by pyoverdin. Analogous to FecA, FpvA contains an

amino-terminal extension; deletion of the extension

abolishes induction of pyoverdin synthesis in response

to pyoverdin in the growth medium but retains pyoverdin

transport [12]. PvdS binds to the RNA polymerase core

enzyme in a 1:1 ratio and this complex binds to a DNA

promoter fragment of the pvdA pyoverdin biosynthesis

gene [13�]. The proposed transcription model is similar to

the Fec transcription model. Fe3þ pyoverdin binds to the

FpvA outer-membrane transport protein that interacts

with FpvR. The signal is transmitted across the cytoplas-

mic membrane via the predicted FecR transmembrane

segment, and then PvdS dissociates from FpvR and

functions as an ECF s factor. A second fecI homologue,

fpvI, maps adjacent to fpvR but is transcribed in the

opposite direction. FpvI also receives a transcription

initiation signal from FpvR.

In a study demonstrating interactions of FecI and FecR in

E. coli, two pairs of FecIR homologues of P. aeruginosawere included [9]. As demonstrated by the LexA two-

hybrid system, the FecI homologue PA2468 (Figure 1)

and its truncated form PA2468110–172 dimerise with the

related FecR1–85 homologue PA24671–90 but not with the

unrelated FecR1–85 homologue PA39001–85 [9]. PA39001–85

only dimerises with the related FecI homologue PA3899

(Figure 1) and the truncated PA3899105–170. The trun-

cated FecI-like fragments cover region 4, demonstrating

their involvement in the interaction with the FecR homo-

logues of P. aeruginosa. As with fecIR, the fecIR-like

Pseudomonas genes are preceded by Fur boxes. In addi-

tion, genome analysis of P. aeruginosa reveals 14 ECF sfactors of the fecI type, which are adjacent to fecR-type

regulatory genes. Ten of these fecIR-like genes are adja-

cent to fecA-like genes that encode proteins with amino-

terminal extensions like FecA [14��].

Regulatory systems analogous to FecAIR have been

identified experimentally in Bordetella pertussis [15], Bor-detella bronchiseptica [16] and in Bordetella avium [17]

(Figure 5). They all regulate iron transport systems for

which the iron ligand responsible for induction or regula-

tion by iron has been shown. B. pertussis and B. bronch-iseptica encode the two regulatory genes hurI and hurRupstream of the heam transport gene cluster bhuRSTUV.

Synthesis of the BhuR outer-membrane transport protein

for haem is enhanced when cells are grown in a medium

supplemented with hemin [15]. In addition, synthesis of a

putative ferric siderophore outer-membrane transport

protein (BfrZ) of B. bronchiseptica is regulated by two

proteins, BupI and BupR, which are homologous to the

fecI and fecR gene products, respectively [16]. Overexpres-

sion of bupI induces bfrZ transcription and BfrZ is

observed in the outer-membrane fraction. B. avium con-

tains a haem utilisation system in which the synthesis of

the related outer-membrane transport protein BhuR is

induced by haem and requires RhuI (which is homolo-

gous to FecI). BhuR synthesis is enhanced in cells that

overexpress RhuI. Overexpression of RhuI reduces tran-

scription of a ss-dependent gene, suggesting competition

Figure 5

fpvI

fecA

bhuR

pupB

bhuR

bfrZ

prhA

fecB

bhuS

fecC

bhuT

fecD

bhuU

fecE

bhuV

fecI

hurI

rhuI

bupI

prhI

pupI

fecR

hurR

rhuR

bupR

prhR

pupR

fpvR fpvA pvdS


Comparison of the arrangement of the fecIR and fecABCDE genes of E.

coli K-12 with similar transport and signalling systems. The arrows

indicate the transcription polarity of the genes. (See text for designation

of the genes.)

178 Cell regulation


between RhuI and ss for the RNA polymerase core

enzyme [17].

Ralstonia solanacearum elicits a hypersensitive response

on non-host plants. At the beginning of the regulatory

cascade that eventually induces the hrp hypersensitivity

genes stands a regulatory device of the Fec type

[18,19,20�]. In the R. solanacearum system, it is highly

interesting that the initial signal is generated by physical

contact between the bacteria and the plant cells, without

involvement of a diffusible substance. A signalling system

that starts at the bacterial cell surface is perfectly suited to

respond to cell–cell contact, as was previously predicted

[2]. For the expression of the prhJ regulatory gene, the

PrhA outer-membrane protein, PrhI (homologous to

FecI) and PrhR (homologous to FecR) are required. As

with the fecIR genes, the prhIR genes form a separate

regulatory unit (Figure 5) that does not require PrhA for

expression and is not autoregulated. In addition, cells

synthesising a truncated PrhR protein are fully patho-

genic on host plants and this is reminiscent of the con-

stitutive expression of the fec transport genes in cells

synthesising truncated FecR proteins.

ConclusionsSince its identification in 1994, the ECF s factor family

has rapidly grown. In the FecIR subgroup, all studied

systems except one regulate the synthesis of Fe3þ trans-

port systems. The reason might be found in the very low

Fe3þ availability, which requires intricate transport sys-

tems across the outer membrane, through the periplasm,

and across the cytoplasmic membrane. Control that starts

from the cell surface enables transcription initiation with-

out formation of the transport system in the cytoplasmic

membrane. It is economic to synthesise the iron-import

systems only when they are needed. Iron starvation alone

is not a sufficient signal for the formation of the transport

systems, since a single strain may express several iron

transport systems. For example, E. coli has up to eight

such systems, and only one system transports the source

of iron that may be available at a given time.

An additional regulatory system that confers specificity to

the available iron source limits the metabolic activity of

the cells to what is required. This is the case for the

regulation of the ferric citrate transport system. Iron

starvation induces synthesis of the regulatory proteins

FecI and FecR, of which only a few molecules are

synthesised. When ferric citrate is in the medium, synth-

esis of FecA is strongly increased from a basal level to

around 80,000 molecules per cell, making FecA one of the

most highly synthesised proteins of E. coli. The high

levels of FecA facilitate capture of the scarce (Fe3þ

citrate)2 molecules in the medium. Binding of (Fe3þ

citrate)2 to FecA at the cell surface initiates a signal that

eventually ends in the cytoplasm, where transcription of

the fec transport genes is initiated. FecR is required for

transmission of the signal through the periplasm and

across the cytoplasmic membrane to convert FecI to an

active s factor. As FecI shows virtually no activity in the

absence of FecR, FecR cannot function purely as an anti-sfactor. If FecR is an anti-s factor, which is likely in the

light of the regulation of other ECF s factors, FecR must

display an additional function — for example, to maintain

FecI in an active conformation.

The conformational change in FecR that results from the

signal received from FecA occupied with (Fe3þ citrate)2

might cause dissociation of FecI from FecR, which then

immediately binds to the RNA polymerase core enzyme

to avoid inactivation by proteolysis, denaturation or pre-

cipitation. FecR might also activate FecI by changing the

FecI conformation. There is no evidence for a chemical

modification of FecI, however. It is also difficult to

envisage that FecR catalyses chemical modification of

FecI, because the smallest fragment of FecR that acti-

vates FecI is FecR1–68 [2], which is unlikely to display

enzymatic activity. Without a signal, the cytoplasmic

fragment of FecR, which shows constitutive expression

of the fec transport genes in the absence of inducer, might

activate FecI by spontaneous dissociation from FecI or by

spontaneous induction of the active conformation in FecI.

The few studies on other ECF s factors of the FecI type

suggest a mechanism similar to the FecAIR signalling

cascade. Bacterial species living in environments of poor

and frequently changing nutrients such as soil and water

are particularly rich in ECF s factors. They are also rich in

TonB-dependent outer-membrane transport proteins that

indicate that scarce nutrients, other than iron, are taken up

by active-transport systems across the outer membrane.

These transport proteins might not only function as trans-

porters but also as signalling proteins for ECF s factors.

All the systems discussed in this review concern regula-

tion of virulence factors of pathogenic or potentially

pathogenic bacteria. Regulation of the genes that control

the hypersensitivity response in plants by R. solana-cearum, and exoproteinase and exotoxin synthesis in

P. aeruginosa, extends the signals and the functions that

are controlled by the FecIRA-type regulation beyond

regulation of iron-transport systems. Following the dis-

covery of signal generation by cell–cell contact in the

R. solanacearum–plant system, it will be of interest to see

if a similar regulatory device functions during infection of

animals or humans — for example, in the regulation of

the formation of type III and type IV secretion systems

that are induced upon contact of bacteria with eukaryotic

cells of their hosts.

AcknowledgementsThe authors’ work was supported by the Deutsche Forschungsgemeinschaft(Br 330/19-1), the Alexander von Humboldt Foundation (M Ogierman) andthe Fonds der Chemischen Industrie.



References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest��of outstanding interest

1. Lonetto MA, Brown KL, Rudd KE, Buttner MJ: Analysis of theStreptomyces coelicolor sigE gene reveals the existence of asubfamily of eubacterial RNA polymerase r factors involved inthe regulation of extracytoplasmic functions. Proc Natl Acad SciUSA 1994, 91:7573-7577.

2. Braun V: Surface signaling: novel transcription initiationmechanism starting from the cell surface. Arch Microbiol 1997,167:325-331.

3. Luck SN, Turner SA, Rajakumar K, Sakellaris H, Adler B: Ferricdicitrate transport system (FEC) of Shigella flexneri 2a YSH6000is encoded on a novel pathogenicity island carrying multipleantibiotic resistance genes. Infect Immun 2001, 69:6012-6021.

4. Maeda H, Jishage M, Nomura T, Fujita N, Ishihama A: Twoextracytoplasmic function sigma subunits rE and rFecI ofEscherichia coli: promoter selectivity and intracellular levels.J Bacteriol 2000, 182:1181-1184.

5.��

Ferguson AD, Chakraborty R, Smith BS, Esser L, van der Helm D,Deisenhofer J: Structural basis of gating by the outer membranetransporter FecA. Science 2002, 295:1715-1719.

This paper describes the crystal structure of FecA in the unloaded andferric-citrate-loaded conformation. It also reveals substrate gating, andforms the basis for the interpretation of the properties of FecA mutantproteins.

6. Kim I, Stiefel A, Plantor S, Angerer A, Braun V: Transcriptioninduction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton systemand the electrochemical potential of the cytoplasmicmembrane. Mol Microbiol 1997, 23:333-344.

7. Welz D, Braun V: Ferric citrate transport of Escherichia coli:functional regions of the FecR transmembrane regulatoryprotein. J Bacteriol 1998, 180:2387-2394.

8. Enz S, Mahren S, Stroeher UH, Braun V: Surface signaling in ferriccitrate transport gene induction: interaction of the FecA, FecRand FecI regulatory proteins. J Bacteriol 2000, 182:637-646.

9. Mahren S, Enz S, Braun V: Functional interaction of region 4 ofthe extracytoplasmic function sigma factor FecI with thecytoplasmic portion of the FecR transmembrane protein of theEscherichia coli ferric citrate transport system. J Bacteriol 2002,184:3704-3711.

10. Koster M, van Klompenburg W, Bitter W, Leong J, Weisbeek P:Role for the outer membrane ferric siderophore receptor PupBin signal transduction across the bacterial cell envelope.EMBO J 1994, 13:2805-2813.

11. Lamont IL, Beare PA, Ochsner U, Vasl AI, Vasil ML: Siderophore-mediated signaling regulates virulence factor production inPseudomonas aeruginosa. Proc Natl Acad Sci USA 2002,99:7072-7077.

12. Leoni L, Orsi N, De Lorenzo V, Visca P: Functional analysis ofPvdS, an iron starvation sigma factor of Pseudomonasaeruginosa. J Bacteriol 2000, 182:1481-1491.

13.�

Shen J, Meldrum A, Poole K: FpvA receptor involvement inpyoverdine biosynthesis in Pseudomonas aeruginosa.J Bacteriol 2002, 184:3268-3275.

This paper provides biochemical evidence that FpvA acts as a transcrip-tion regulatory protein and as an outer-membrane transporter.

14.��

Visca P, Leoni L, Wilson MJ, Lamont IL: Iron transport andregulation, cell signalling and genomics: lessons fromEscherichia coli and Pseudomonas. Mol Microbiol 2002,45:1177-1190.

This paper provides a comprehensive overview of regulatory devices ofthe FecIR type in P. aeruginosa.

15. Vanderpool CK, Armstrong SK: The Bordetella bhu locus isrequired for heme iron utilization. J Bacteriol 2001,183:4278-4287.

16. Pradel E, Locht C: Expression of the siderophore receptor genebfrZ is controlled by the extracytoplasmic-function sigmafactor BupI in Bordetella bronchiseptica. J Bacteriol 2001,183:2910-2917.

17. Kirby AE, Metzger DJ, Murphy ER, Connell TD: Heme utilization inBordetella avium is regulated by RhuI, a heme-responsiveextracytoplasmic function sigma factor. Infect Immun 2001,69:6951-6961.

18. Marenda M, Brito B, Callard D, Genin S, Barberis P, Boucher C,Ariat M: PrhA controls a novel regulatory pathway required forthe specific induction of Ralstonia solanacearum hrp genes inthe presence of plant cells. Mol Microbiol 1998, 27:437-453.

19. Aldon D, Brito B, Boucher C, Genin S: A bacterial sensor of plantcell contact controls the transcriptional induction of Ralstoniasolanacearum pathogenicity genes. EMBO J 2000,19:2304-2314.

20.�

Brito B, Aldon D, Barberis P, Boucher C, Genin S: A signal transfersystem through three compartments transduces the plant cellcontact-dependent signal controlling Ralstonia solanacearumhrp genes. Mol Plant Microbiol 2002, 15:109-119.

This paper shows that ECF sigma-factor-controlled transcription initiationoccurs at the start of a signal cascade that regulates the hypersensitivityresponse.

21. Moeck GS, Letellier L: Characterization of in vitro interactionsbetween a truncated TonB protein from Escherichia coli andthe outer membrane receptors FhuA and FepA. J Bacteriol 2001,183:2755-2764.

22. Higgs PI, Larsen RA, Postle K: Quantification of knowncomponents of the Escherichia coli TonB energy transductionsystem: TonB, ExbB, ExbD and FepA. Mol Micobiol 2002,44:271-281.

23. Stiefel A, Mahren S, Ochs M, Schindler PT, Enz S, Braun V: Controlof the ferric citrate transport system of Escherichia coli:mutations in region 2.1 of the FecI extracytoplasmic-functionsigma factor suppress mutations in the FecR transmembraneregulatory protein. J Bacteriol 2001, 183:162-170.

180 Cell regulation


Documents

Fec1 and Sigma Factor