# Identification of IbeR as a Stationary-Phase Regulator in MeningiticEscherichia coli K1 that Carries a Loss-of-Function Mutation in rpoS

Authors: Feng Chi; Ying Wang; Timothy K. Gallaher; Chun-Hua Wu; Ambrose Jong; Sheng-He Huang
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation.


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## Body

## 1. Introduction

Neonatal bacterial meningitis continues to be
the most common serious infection of the central nervous system (CNS) in
newborns with high morbidity and mortality despite the availability of
effective bactericidal antibiotics over the last sixty years [1, 2]. This high morbidity and mortality are due to inadequate
knowledge of the pathogenesis of this disease.E. coli is the most common gram-negative
bacterium causing neonatal sepsis and meningitis [1]. Bacterial meningitis in
newborns is due to hematogenous spread of the pathogen to the meninges. The
most important issue in the pathogenesis of E.
coli meningitis is how circulating pathogens cross the blood-brain barrier
(BBB), which is mainly composed of brain microvascular endothelial cells (BMECs)  [3, 4]. Our
previous studies showed that E. coli K1 invasion of human BMEC was
significantly greater with stationary-phase (SP) cultures than with
exponential-phase cultures, suggesting that expression of E. coli K1 invasion-associated virulence
genes is strongly regulated in a growth-phase-dependent manner [5]. A nonsense mutation in the SP regulatory gene rpoS was identified in E. coli K1 strains E44 and IHE3034 [5].
Complementation with the wild type E. coli K12 rpoS gene
significantly enhanced IHE3034 invasion of BMEC, but failed to improve the invasion activity of another E.
coli K1 strain E44. These studies suggest that the growth-phase-dependent
invasion of BMEC by IHE3034 is affected by RpoS and that E44 carries a
loss-of-function mutation in the rpoS gene. However, the SP gene
regulation in E44 has remained an unanswered question.Several virulence
factors, including Ibe (termed afterinvasion of  brainendothelial
cells) proteins [6, 7], OmpA  [8], YiijP [9], FimH [13], AslA [14], and TraJ
[15], have been identified in various strains of E. coli in the in vitro and in vivo models of the
BBB as invasins. Most of those
invasion genes are present in the E. coli K-12 genome [4, 16]. However, the ibeA gene encoding a 50 kDa protein has been found to be unique to some pathogenic E. coli K1 strains (e.g., C5 and RS218),
while laboratory strains of E. coli K-12 (e.g., DH5 and HB101), as well
as noninvasive E.  coli (e.g., E412),
lack ibeA [4]. Recently, vimentin has
been identified as an IbeA-binding protein on the surface of human BMEC [17].Using theibeA gene
as a probe, we have identified a 20.3 kb genomic locus as a geneticisland of meningiticE. coli containing ibeA (GimA) [16]. This
locus is situated between yjiD and yjiE, adjacent to the fim operon, and
absent in nonpathogenic E. coli K12
strains. GimA consists of 15 genes that form 4 operons. The first three operons
(ptnIPKC, cglDTEC, gcxKRCI) may
be involved in energy metabolism and the last operon (ibeRAT) contributes to E.
coli K1 invasion of BMEC. Our previous work showed that GimA-mediated
invasion of human endothelial cells is regulated by carbon source [16]. This is
consistent with the observations by others that carbon source modulates
expression of virulence factors in several pathogenic bacteria [4]. The ibeRAT operon encodes IbeR, IbeA, and IbeT. IbeA and IbeT contribute
to E. coli K1 invasion and adhesion
[18, 19]. Our previous studies suggest that IbeR is a novel regulatory protein that is present in pathogenic E. coli K1 [16]. It belongs to the
NtrC/NifA family of transcriptional activators, carrying a sigma 54-interaction
domain and showing significant
sequence homology to various regulatory proteins for glycerol metabolism operon
in Citrobacter freundii (P45512), acetoacetate metabolism in E. coli K12,
sigma L-dependent transcription in B.
subtilis (P54529), NIF-specific regulation in Herbaspirillum seropedicae, dhaR transcription in E. coli K12, and globe signal transduction
in Clostridium beijerinckii [4, 20]. However, it is unknown how
IbeR contributes to the pathogenesis of meningitic infection by modulating the
virulence of the pathogen. As E44 carries a nonsense mutation in the rpoS gene and exhibits strong invasion activity in the SP, there
must be alternative regulatory mechanisms responsible for the SP gene
expression. We speculated that ibeR is an rpoS-like regulator in SP gene expression in E44. In order to dissect the
regulation of SP gene expression in E44 that is associated with the
pathogenesis of E. coli meningitis, a comparative proteomic analysis of
an ibeR deletion mutant (BR2) and its parent strain E44 was carried out
in this study. Our studies suggested that the ibeR gene was involved in
regulating SP gene expression related to stress resistance and pathogenesis.


## 2. Materials and Methods

### 2.1. Bacterial Strains, Plasmids, and Medium

The bacterial strain and plasmid vectors and their relevant
characteristics are shown in Table1. The mutant strains used in this study
were derived from E44, which is a rifampin-resistant strain derived from a
neonatal meningitis isolate, E. coli K1 RS218 (O18:K1:H7)  [4, 21]. E. coli DH5α and pGEM-T easy vector were
used for subcloning. SM10 (λpir), DH5α  (λpir), and pCVD442 were used for making
isogenic deletion mutants of ibeR and tnaA [6, 10, 11]. E.
coli K12 strain MC4100 [22] and its rpoS
insertion mutationTn10 mutant RH90 [23]
were used as positive and negative controls for RpoS, respectively. Plasmid
pStyABB, which carries the gene for monooxygenase, was used for indole assay
[12]. E. coli strains were grown at 37°C in Luria broth (LB; 1%
tryptone, 0.5% yeast extract, 0.5% NaCl) or brain heart infusion (BHI, Difco
Laboratories, Detroit, Mich, USA) broth and were stored at  −70°C in LB plus 20%
glycerol. When it was necessary, the medium was supplemented with ampicillin
(100 μg/mL) and rifampin (100 μg/mL) for the positive selection of plasmids or
bacterial strains (Table 1).Table 1
E. coli K1 (meningitic) or K12
(nonpathogenic) strains and plasmids used in this study.
Strain or plasmidCharacteristicsReference(s)StrainsRS218O18:K1:H7 (CSF)[6–9]E44RS218, Rifr[6–9]DH5α  (λpir)K 12 strain[6, 7]SM10 (λpir)K 12 strain[6, 7]MC4100K 12 strain[5]RH90K 12 strain,rpoS deletion mutant[5]BR2ibeR deletion
mutant of E44This studyTNA44tnaA deletion
mutant of E44This studyPlasmidspCVD442Ampr, oiRr6K, sacB, mobRP4[10, 11]pCBR2pCVD442 carrying anibeR deletion, AmprThis studypGEM-TAmpr, lacZPromegapCTNA2pCVD442 carrying atnaA deletion, AmprThis studypGTNApGEM-T carrying a 3.7 kb fragment containingtnaA geneThis studypStyABBcontaining the gene of monooxygenase for indole
assay, Ampr[12]pWKS30Ampr, lacZ[6]pWKS1030pWKS30 carrying an 18 KbibeR locus[6]Ampr, ampicillin resistant; lacZ,
a partial gene coding for the N-terminal fragment of β-galactosidase; Kanr,
kanamycin resistant; Rifr, rifampin resistant.


### 2.2. Extraction and Manipulation of Plasmids and Subcloning

All genetic manipulations were done by using standard
methods, as described elsewhere [24]. Plasmid DNA was extracted by using a
plasmid mini kit (Qiagen, Calif,
USA). DNA fragments were purified and were extracted from agarose
gel slices, using QIAquick Gel Extraction Kit (Qiagen). Competent E. coli cells were made in 10% glycerol
and were transformed by electroporation as described previously [6, 7].


### 2.3. Construction of Isogenic in-Frame Deletion Mutants ofibeR and tnaA

To determine the role of theibeR gene in the
growth-phase-dependent E. coli K1 invasion of BMEC, an isogenic deletion
mutant of ibeR was generated as
follows. Two PCR DNA fragments, B (1.2 kb) and R (1.0 kb), flanking a 1.8-kb
region to be deleted were produced from two pairs of primers (IbeR-S1/IbeR-B1
for B and IbeR-B2/IbeR-X2 for R, see Table 2). The two fragments were ligated
to make a 2.2 kb fragment (BR) that carries an ibeR internal deletion.
The BR fragment was subcloned between SalI and XbaI sites on pCVD442 [10], and
the resulting recombinant plasmid was named pCBR2. The mutants named BR2 were
obtained by mating E44 with SM10 λpir that carries pCBR2 as described previously
[6]. We used PCR and DNA sequencing to confirm the internal deletion in the ibeR deletion mutant BR2 and the desired
chromosomal gene ibeR of the mutant with primers IbeR-S1 and IbeR-X2
(Table 2). Amplification was done by using the following cycle profile: 35
cycles at 94°C for 1 minute, 58°C for 1 minute, and 70°C for 1.5 minutes.Table 2
Oligonucleotides used for cloning,
sequencing, and making the deletion mutants ofibeR and tnaA genes.
PrimersSequences of primersRetained amino acidsprimers foribeR deletionTotal  56
residuesIbeR-S1
(S = SalI)5′-GATGTCGACGGGCTTTTCGGCGTCA-3′52 N-terminal
residuesIbeR-B1
(B = BamHI)5′–CGGGATCCAGTGGCGAGGGTCACA-3′(MDIIIMNKES...)IbeR-B2
(B = BamHI)5′-CAGGATCCAAATGTTGAGCATGCAG-3′4 C-terminal
residuesIbeR-X2
(X = XbaI)5′-CGTCTAGATAAGGGCTAAACATATCG-3′(...GSKC)primers fortnaA  deletionTotal  56  residuesTN-S1
(S = SalI)5′-GGGTCGACCAGAGATCTGGCCGGAAT
T-3′21 N-terminal
residuesTN-B1
(B = BamHI)5′-ACGGATCCAATAACACGAATGCGGAACGGTTC-3′(MKDYVMENFK...)TN-B2
(B = BamHI)5′-TTAGATCTTTTAAACATGTGAAAGAGAACGCG-3′35 C-terminal residuesTN-X2
(X = XbaI)5′-CCTCTAGATTAGCCAAATTTAGGTAACAC
G-3′(...RHFTAKLKEV)For thetnaA in-frame deletion mutant, the same method
was used as ibeR deletion. Briefly, 2 PCR DNA fragments, FTN5 (1.0-kb)
and FTN3 (1.4-kb), were made to flank a 1.3 kb region containing tnaA to
be deleted, by using 2 primer pairs (TN-S1/TN-B1 for FTN5 and TN-B2/TN-X2 for
FTN3, see Table 2). Then the two fragments were ligated to make a 2.4 kb
fragment (FTN53) that carried a tnaA internal deletion. The FTN53
fragment was subcloned into pCVD442 between SalI and XbaI sites to get the
suicide plasmid pCTNA2. To get the tnaA in-frame deletion mutant, TNA44,
conjugation and screening were carried out as described above. The tnaA gene deletion in the mutant TNA44
was confirmed by PCR using the primers TN-S1 and TN-X2 (Table 2).


### 2.4. Invasion Assay

Human BMECs were routinely cultured in RPMI 1640 medium
(Mediatech, Herndon, Va,
USA) containing 10% heat inactivated fetal bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM sodium pyruvate, essential amino acids, vitamins, penicillin
G (50μg/mL), and streptomycin (100 μg/mL) at 37°C in 5% CO2 [7].
Invasion assays were performed as previously described [6, 7, 18]. The
number of intracellular bacteria was determined on blood agar plates after the
extracellular bacteria were killed by incubation of the monolayers with
experimental medium containing gentamicin (100 μg/mL) for 1 hour. Results were
expressed as percent invasion (100 × (number of intracellular bacteria
recovered)/(number of bacteria inoculated)).


### 2.5. Protein Extraction and 2DE

Protein extraction
and 2DE were carried out as described [25] with minor modifications. Briefly, E.
coli strain E44 and the ibeR deletion mutant BR2 were cultured
in BHI medium overnight without agitation. The bacterial cells were harvested
at the stationary-phase (OD = 2.5−3.0) by
centrifugation at 6000 × g for 10 minutes. Denaturing protein extraction (phenol
extraction procedure) was performed according to Saravanan and Rose [26]. The lyophilized pellets were
dissolved in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2% pH 3–10 Biolytes, 1%
DTT; 1 mg dry pellets for 200 μL buffer) and shaken on vortex for 1 hour at
room temperature. The first and second dimensions of the PAGE were performed at
least in triplicate (to reduce the likelihood of differences based solely upon
gel-to-gel variability) according to the standard protocols developed and
defined by Bio-Rad. Solubilized total E.
coli protein samples (200 μL each) were loaded on 11 cm immobilized pH
gradient (IPG) strips (pH 4–7).
Rehydration/loading was done passively (no voltage) for 1 hour, followed
immediately by 14 hours of active rehydration (50 V) at 20°C. Isoelectric
focusing of the IPG strips was performed at 20°C using a 50 μA  current limit
per strip to prevent damage to the strip and the instrument. Electrophoresis
was carried out as follows: step 1, 250 V for 20 minutes; step 2, a rapid ramp
to 8000 V; step 3,
focusing at 8000 V for 55 000 Vhr; step 4,
hold at 400 V. Due to latent ionic components in the sample the actual running
voltage was only approximately 6500 V. After the first dimensional run was completed, the IPG
strips were equilibrated in buffer I (6 M urea, 0.375 M Tris-HCl pH 8.8, 2% SDS,
20% glycerol, and 2%DTT) for 15 minutes and then in buffer II (6 M urea, 0.375 M
Tris-HCl pH 8.8, 2% SDS, 20% glycerol, and 2%DTT) for additional 15 minutes.
The second dimension SDS-PAGE was performed with 15% resolving gels and 5%
stacking gels (160 × 180 × 0.5 mm). The gels were stained with 0.1% coomassie brilliant
blue (CBB) R-250. The 2DE gels were scanned at a 200 bpi resolution with
Typhoon scanner (Amersham Biosciences, NJ, USA), and analyzed with ImageMaster
2D Platinum version 6.0 (GE Healthcare BIO-Science, NJ, USA). Only those protein spots having
differences in density of 1.5-fold or greater between the groups were chosen.
Moreover, all protein spots selected for analysis were shown to have
significant difference in protein density (mean ± SEM, P<.01) by software of SPSS 10.0.


### 2.6. In-Gel Protein Digestion

Protein bands were excised from preparative coomassie blue-stained
gels and washed several times with destaining solutions (25 mM NH4HCO3 for 15 minutes and then with 50% acetonitrile containing 25 mM NH4HCO3 for 15 minutes). Gel pieces were then dehydrated with 100% acetonitrile, dried,
and then incubated with a reducing solution (25 mM NH4HCO3 containing 10 mM dithiothreitol) for 1 hour at 56°C and subsequently with an
alkylating solution (25 mM NH4HCO3 containing 55 mM
iodoacetamide) for 45 minutes at room temperature. After reduction and
alkylation, gels were washed several times with the destaining solutions and
finally with pure water for 15 minutes before being treated again with 100% acetonitrile.
Depending on the protein content, 2-3 μL of 0.1 μg/μL modified
trypsin (Promega, Wiss, USA,
sequencing grade) in 25 mM NH4HCO3 was added over the gel
spots and incubated for 30 minutes. About 7–10 μL of 25 mM NH4HCO3 was then added to cover the gel spots and incubated at 37°C overnight. The
in-gel digestion products were extracted with formic acid/acetonitrile
solutions followed by evaporation. Samples were desalted using mZipTip C18
pipette tips (Millipore, Mass, USA) before MS/MS analysis.


### 2.7. Protein Identification by LC-MS/MS

The sample was
resuspended in 10μL of 0.1% formic acid, injected via an autosampler (Surveyor,
ThermoFinnigan, Calif,
USA) and subjected to reverse phase liquid chromatography using ThermoFinnigan
Surveyor MS-Pump in conjunction with a BioBasic 18 100 × 0.18 mm reverse-phase
capillary column (ThermoFinnigan, Calif, USA). Mass analysis was done using a
ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with a
nanospray ion source (ThermoFinnigan, Calif, USA) employing a 4 cm metal emitter (Proxeon,
Odense, Denmark). Spray voltage of the mass spectrometer was set to 2.9 kV and capillary temperature was set at 190°C. The column
equilibrated for 5 minutes at 1.5 μL/min with 95% Solution A and 5% Solution B
(A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile) followed by
a linear gradient was initiated 5 minutes after sample injection ramping to 65%
Solution A over 45 minutes. Solution A was increased to 80% over the
subsequent 5 minutes and held at 80% for 5 minutes, after which the column was
reequilibrated back to 5% Solution A (aqueous). Mass spectra were acquired in
the m/z 400–1800 range. A
data-dependent acquisition mode was used where each of the top five ions for a
given scan was subjected to MS/MS analysis. The protein identification was conducted with the MS/MS
search software Mascot 1.9 with confirmatory or complementary analyses using
TurboSequest as implemented in the Bioworks 3.2. E. coli genome
sequences at the National Center for Biotechnology Information (NCBI) were used
as the primary search databases and searches were complemented with the NCBI
nonredundant protein database.


### 2.8. Indole Assay

For
determination of indole production, we followed the method of indole conversion
into indigo as described previously [12, 27], with minor modifications. All
strains E44, BR2, TNA44, MG1655, and RH90 were transformed with the pStyABB
plasmid, which constitutively expresses the StyAB protein converting indole to
indigo. The bacteria were incubated in M9 medium containing 0.4% glucose and
100 μg/mL ampicillin overnight with shaking. Then the bacteria were collected by
centrifugation and resuspended to OD600 = 0.2 in BHI medium supplied
with 100 μg/mL ampicillin, and incubated at 37°C without shaking. To determine
the indigo formation at different time points, batch-grown cells were harvested
every 2 hours by centrifugation. Then the bacteria were lysed in DMSO for 30
minutes. The samples were read at 600 nm to determine the indigo concentration
by comparison to a standard curve.


### 2.9. Determination of Resistance to Environmental Stress

Bacteria were grown in BHI broth at 37°C overnight without
shaking, and collected by centrifugation. The number of cells was measured on
the basis of their OD at 600 nm. Bacteria were suspended and diluted to 107 cells/mL in PBS for the following assays. For heat shock, 100 μL of bacteria
was heated at 54°C for 3 minutes. For alkali endurance, the bacterial
suspension was mixed with equal volume of Tris buffer (1 M, pH = 10.0) and 8
volumes of water (final concentration, 100 mM, pH 10.0) and incubated at 37°C
for 30 minutes. For acid endurance, 1/10 volume of the bacterial suspension was
mixed with LB containing acetic acid (final concentration, 90 mM, pH 2.8) and
incubated at 37°C for 20 minutes. For high osmolarity challenge, bacteria were
mixed with an equal volume of 4.8 M NaCl (final concentration, 2.4 M) and
incubated at 37°C for 1 hour. For oxidative stress, bacteria were harvested and
resuspended in an equal volume of PBS containing 10 μM H2O2 incubated at 37°C  for 30 minutes. After exposure to these stresses, bacteria
were diluted in 0.9% saline and plated in duplicate on LB agar plates. The surviving rate with stress was
calculated from the ratio of the bacterial number under stress condition to the
bacteria number under nonstress condition. The surviving rate without stress was
calculated from the bacteria number grown on plates.


## 2.1. Bacterial Strains, Plasmids, and Medium

The bacterial strain and plasmid vectors and their relevant
characteristics are shown in Table1. The mutant strains used in this study
were derived from E44, which is a rifampin-resistant strain derived from a
neonatal meningitis isolate, E. coli K1 RS218 (O18:K1:H7)  [4, 21]. E. coli DH5α and pGEM-T easy vector were
used for subcloning. SM10 (λpir), DH5α  (λpir), and pCVD442 were used for making
isogenic deletion mutants of ibeR and tnaA [6, 10, 11]. E.
coli K12 strain MC4100 [22] and its rpoS
insertion mutationTn10 mutant RH90 [23]
were used as positive and negative controls for RpoS, respectively. Plasmid
pStyABB, which carries the gene for monooxygenase, was used for indole assay
[12]. E. coli strains were grown at 37°C in Luria broth (LB; 1%
tryptone, 0.5% yeast extract, 0.5% NaCl) or brain heart infusion (BHI, Difco
Laboratories, Detroit, Mich, USA) broth and were stored at  −70°C in LB plus 20%
glycerol. When it was necessary, the medium was supplemented with ampicillin
(100 μg/mL) and rifampin (100 μg/mL) for the positive selection of plasmids or
bacterial strains (Table 1).Table 1
E. coli K1 (meningitic) or K12
(nonpathogenic) strains and plasmids used in this study.
Strain or plasmidCharacteristicsReference(s)StrainsRS218O18:K1:H7 (CSF)[6–9]E44RS218, Rifr[6–9]DH5α  (λpir)K 12 strain[6, 7]SM10 (λpir)K 12 strain[6, 7]MC4100K 12 strain[5]RH90K 12 strain,rpoS deletion mutant[5]BR2ibeR deletion
mutant of E44This studyTNA44tnaA deletion
mutant of E44This studyPlasmidspCVD442Ampr, oiRr6K, sacB, mobRP4[10, 11]pCBR2pCVD442 carrying anibeR deletion, AmprThis studypGEM-TAmpr, lacZPromegapCTNA2pCVD442 carrying atnaA deletion, AmprThis studypGTNApGEM-T carrying a 3.7 kb fragment containingtnaA geneThis studypStyABBcontaining the gene of monooxygenase for indole
assay, Ampr[12]pWKS30Ampr, lacZ[6]pWKS1030pWKS30 carrying an 18 KbibeR locus[6]Ampr, ampicillin resistant; lacZ,
a partial gene coding for the N-terminal fragment of β-galactosidase; Kanr,
kanamycin resistant; Rifr, rifampin resistant.


## 2.2. Extraction and Manipulation of Plasmids and Subcloning

All genetic manipulations were done by using standard
methods, as described elsewhere [24]. Plasmid DNA was extracted by using a
plasmid mini kit (Qiagen, Calif,
USA). DNA fragments were purified and were extracted from agarose
gel slices, using QIAquick Gel Extraction Kit (Qiagen). Competent E. coli cells were made in 10% glycerol
and were transformed by electroporation as described previously [6, 7].


## 2.3. Construction of Isogenic in-Frame Deletion Mutants ofibeR and tnaA

To determine the role of theibeR gene in the
growth-phase-dependent E. coli K1 invasion of BMEC, an isogenic deletion
mutant of ibeR was generated as
follows. Two PCR DNA fragments, B (1.2 kb) and R (1.0 kb), flanking a 1.8-kb
region to be deleted were produced from two pairs of primers (IbeR-S1/IbeR-B1
for B and IbeR-B2/IbeR-X2 for R, see Table 2). The two fragments were ligated
to make a 2.2 kb fragment (BR) that carries an ibeR internal deletion.
The BR fragment was subcloned between SalI and XbaI sites on pCVD442 [10], and
the resulting recombinant plasmid was named pCBR2. The mutants named BR2 were
obtained by mating E44 with SM10 λpir that carries pCBR2 as described previously
[6]. We used PCR and DNA sequencing to confirm the internal deletion in the ibeR deletion mutant BR2 and the desired
chromosomal gene ibeR of the mutant with primers IbeR-S1 and IbeR-X2
(Table 2). Amplification was done by using the following cycle profile: 35
cycles at 94°C for 1 minute, 58°C for 1 minute, and 70°C for 1.5 minutes.Table 2
Oligonucleotides used for cloning,
sequencing, and making the deletion mutants ofibeR and tnaA genes.
PrimersSequences of primersRetained amino acidsprimers foribeR deletionTotal  56
residuesIbeR-S1
(S = SalI)5′-GATGTCGACGGGCTTTTCGGCGTCA-3′52 N-terminal
residuesIbeR-B1
(B = BamHI)5′–CGGGATCCAGTGGCGAGGGTCACA-3′(MDIIIMNKES...)IbeR-B2
(B = BamHI)5′-CAGGATCCAAATGTTGAGCATGCAG-3′4 C-terminal
residuesIbeR-X2
(X = XbaI)5′-CGTCTAGATAAGGGCTAAACATATCG-3′(...GSKC)primers fortnaA  deletionTotal  56  residuesTN-S1
(S = SalI)5′-GGGTCGACCAGAGATCTGGCCGGAAT
T-3′21 N-terminal
residuesTN-B1
(B = BamHI)5′-ACGGATCCAATAACACGAATGCGGAACGGTTC-3′(MKDYVMENFK...)TN-B2
(B = BamHI)5′-TTAGATCTTTTAAACATGTGAAAGAGAACGCG-3′35 C-terminal residuesTN-X2
(X = XbaI)5′-CCTCTAGATTAGCCAAATTTAGGTAACAC
G-3′(...RHFTAKLKEV)For thetnaA in-frame deletion mutant, the same method
was used as ibeR deletion. Briefly, 2 PCR DNA fragments, FTN5 (1.0-kb)
and FTN3 (1.4-kb), were made to flank a 1.3 kb region containing tnaA to
be deleted, by using 2 primer pairs (TN-S1/TN-B1 for FTN5 and TN-B2/TN-X2 for
FTN3, see Table 2). Then the two fragments were ligated to make a 2.4 kb
fragment (FTN53) that carried a tnaA internal deletion. The FTN53
fragment was subcloned into pCVD442 between SalI and XbaI sites to get the
suicide plasmid pCTNA2. To get the tnaA in-frame deletion mutant, TNA44,
conjugation and screening were carried out as described above. The tnaA gene deletion in the mutant TNA44
was confirmed by PCR using the primers TN-S1 and TN-X2 (Table 2).


## 2.4. Invasion Assay

Human BMECs were routinely cultured in RPMI 1640 medium
(Mediatech, Herndon, Va,
USA) containing 10% heat inactivated fetal bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM sodium pyruvate, essential amino acids, vitamins, penicillin
G (50μg/mL), and streptomycin (100 μg/mL) at 37°C in 5% CO2 [7].
Invasion assays were performed as previously described [6, 7, 18]. The
number of intracellular bacteria was determined on blood agar plates after the
extracellular bacteria were killed by incubation of the monolayers with
experimental medium containing gentamicin (100 μg/mL) for 1 hour. Results were
expressed as percent invasion (100 × (number of intracellular bacteria
recovered)/(number of bacteria inoculated)).


## 2.5. Protein Extraction and 2DE

Protein extraction
and 2DE were carried out as described [25] with minor modifications. Briefly, E.
coli strain E44 and the ibeR deletion mutant BR2 were cultured
in BHI medium overnight without agitation. The bacterial cells were harvested
at the stationary-phase (OD = 2.5−3.0) by
centrifugation at 6000 × g for 10 minutes. Denaturing protein extraction (phenol
extraction procedure) was performed according to Saravanan and Rose [26]. The lyophilized pellets were
dissolved in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2% pH 3–10 Biolytes, 1%
DTT; 1 mg dry pellets for 200 μL buffer) and shaken on vortex for 1 hour at
room temperature. The first and second dimensions of the PAGE were performed at
least in triplicate (to reduce the likelihood of differences based solely upon
gel-to-gel variability) according to the standard protocols developed and
defined by Bio-Rad. Solubilized total E.
coli protein samples (200 μL each) were loaded on 11 cm immobilized pH
gradient (IPG) strips (pH 4–7).
Rehydration/loading was done passively (no voltage) for 1 hour, followed
immediately by 14 hours of active rehydration (50 V) at 20°C. Isoelectric
focusing of the IPG strips was performed at 20°C using a 50 μA  current limit
per strip to prevent damage to the strip and the instrument. Electrophoresis
was carried out as follows: step 1, 250 V for 20 minutes; step 2, a rapid ramp
to 8000 V; step 3,
focusing at 8000 V for 55 000 Vhr; step 4,
hold at 400 V. Due to latent ionic components in the sample the actual running
voltage was only approximately 6500 V. After the first dimensional run was completed, the IPG
strips were equilibrated in buffer I (6 M urea, 0.375 M Tris-HCl pH 8.8, 2% SDS,
20% glycerol, and 2%DTT) for 15 minutes and then in buffer II (6 M urea, 0.375 M
Tris-HCl pH 8.8, 2% SDS, 20% glycerol, and 2%DTT) for additional 15 minutes.
The second dimension SDS-PAGE was performed with 15% resolving gels and 5%
stacking gels (160 × 180 × 0.5 mm). The gels were stained with 0.1% coomassie brilliant
blue (CBB) R-250. The 2DE gels were scanned at a 200 bpi resolution with
Typhoon scanner (Amersham Biosciences, NJ, USA), and analyzed with ImageMaster
2D Platinum version 6.0 (GE Healthcare BIO-Science, NJ, USA). Only those protein spots having
differences in density of 1.5-fold or greater between the groups were chosen.
Moreover, all protein spots selected for analysis were shown to have
significant difference in protein density (mean ± SEM, P<.01) by software of SPSS 10.0.


## 2.6. In-Gel Protein Digestion

Protein bands were excised from preparative coomassie blue-stained
gels and washed several times with destaining solutions (25 mM NH4HCO3 for 15 minutes and then with 50% acetonitrile containing 25 mM NH4HCO3 for 15 minutes). Gel pieces were then dehydrated with 100% acetonitrile, dried,
and then incubated with a reducing solution (25 mM NH4HCO3 containing 10 mM dithiothreitol) for 1 hour at 56°C and subsequently with an
alkylating solution (25 mM NH4HCO3 containing 55 mM
iodoacetamide) for 45 minutes at room temperature. After reduction and
alkylation, gels were washed several times with the destaining solutions and
finally with pure water for 15 minutes before being treated again with 100% acetonitrile.
Depending on the protein content, 2-3 μL of 0.1 μg/μL modified
trypsin (Promega, Wiss, USA,
sequencing grade) in 25 mM NH4HCO3 was added over the gel
spots and incubated for 30 minutes. About 7–10 μL of 25 mM NH4HCO3 was then added to cover the gel spots and incubated at 37°C overnight. The
in-gel digestion products were extracted with formic acid/acetonitrile
solutions followed by evaporation. Samples were desalted using mZipTip C18
pipette tips (Millipore, Mass, USA) before MS/MS analysis.


## 2.7. Protein Identification by LC-MS/MS

The sample was
resuspended in 10μL of 0.1% formic acid, injected via an autosampler (Surveyor,
ThermoFinnigan, Calif,
USA) and subjected to reverse phase liquid chromatography using ThermoFinnigan
Surveyor MS-Pump in conjunction with a BioBasic 18 100 × 0.18 mm reverse-phase
capillary column (ThermoFinnigan, Calif, USA). Mass analysis was done using a
ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with a
nanospray ion source (ThermoFinnigan, Calif, USA) employing a 4 cm metal emitter (Proxeon,
Odense, Denmark). Spray voltage of the mass spectrometer was set to 2.9 kV and capillary temperature was set at 190°C. The column
equilibrated for 5 minutes at 1.5 μL/min with 95% Solution A and 5% Solution B
(A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile) followed by
a linear gradient was initiated 5 minutes after sample injection ramping to 65%
Solution A over 45 minutes. Solution A was increased to 80% over the
subsequent 5 minutes and held at 80% for 5 minutes, after which the column was
reequilibrated back to 5% Solution A (aqueous). Mass spectra were acquired in
the m/z 400–1800 range. A
data-dependent acquisition mode was used where each of the top five ions for a
given scan was subjected to MS/MS analysis. The protein identification was conducted with the MS/MS
search software Mascot 1.9 with confirmatory or complementary analyses using
TurboSequest as implemented in the Bioworks 3.2. E. coli genome
sequences at the National Center for Biotechnology Information (NCBI) were used
as the primary search databases and searches were complemented with the NCBI
nonredundant protein database.


## 2.8. Indole Assay

For
determination of indole production, we followed the method of indole conversion
into indigo as described previously [12, 27], with minor modifications. All
strains E44, BR2, TNA44, MG1655, and RH90 were transformed with the pStyABB
plasmid, which constitutively expresses the StyAB protein converting indole to
indigo. The bacteria were incubated in M9 medium containing 0.4% glucose and
100 μg/mL ampicillin overnight with shaking. Then the bacteria were collected by
centrifugation and resuspended to OD600 = 0.2 in BHI medium supplied
with 100 μg/mL ampicillin, and incubated at 37°C without shaking. To determine
the indigo formation at different time points, batch-grown cells were harvested
every 2 hours by centrifugation. Then the bacteria were lysed in DMSO for 30
minutes. The samples were read at 600 nm to determine the indigo concentration
by comparison to a standard curve.


## 2.9. Determination of Resistance to Environmental Stress

Bacteria were grown in BHI broth at 37°C overnight without
shaking, and collected by centrifugation. The number of cells was measured on
the basis of their OD at 600 nm. Bacteria were suspended and diluted to 107 cells/mL in PBS for the following assays. For heat shock, 100 μL of bacteria
was heated at 54°C for 3 minutes. For alkali endurance, the bacterial
suspension was mixed with equal volume of Tris buffer (1 M, pH = 10.0) and 8
volumes of water (final concentration, 100 mM, pH 10.0) and incubated at 37°C
for 30 minutes. For acid endurance, 1/10 volume of the bacterial suspension was
mixed with LB containing acetic acid (final concentration, 90 mM, pH 2.8) and
incubated at 37°C for 20 minutes. For high osmolarity challenge, bacteria were
mixed with an equal volume of 4.8 M NaCl (final concentration, 2.4 M) and
incubated at 37°C for 1 hour. For oxidative stress, bacteria were harvested and
resuspended in an equal volume of PBS containing 10 μM H2O2 incubated at 37°C  for 30 minutes. After exposure to these stresses, bacteria
were diluted in 0.9% saline and plated in duplicate on LB agar plates. The surviving rate with stress was
calculated from the ratio of the bacterial number under stress condition to the
bacteria number under nonstress condition. The surviving rate without stress was
calculated from the bacteria number grown on plates.


## 3. Results and Discussions

### 3.1. TheibeR Regulatory Gene Is Required for Invasion of Human BMEC by Meningitic E.   coli K1 Strain E44

The geneibeR in E. coli K1 E44 is predicted as
the only regulatory protein present in the ibeRAT operon in GimA by
bioinformatics approaches [16]. To determine the role of ibeR gene in
the growth-phase-dependent invasion of BMEC by meningitic E. coli K1, an isogenic in-frame deletion mutant of ibeR was
made by chromosomal gene replacement with the recombinant suicide plasmid pCBR2
carrying a 2.2 kb DNA fragment with ibeR internal deletion (Figure 1(a)).
The 2.2 kb DNA fragment was generated by ligation of two PCR amplicons (1.2 and
1.0 kbs) flanking the
1.8 kb ibeR coding region. The ibeR deletion mutant was obtained
by mating E44 with SM10 λpir carrying pCBR2. The mutant colony morphology on LB
agar plates and growth rate in LB broth were the same as the parent strain E44.
The deletion of ibeR was confirmed by colony PCR and DNA sequencing
(Figure 1(b)). In order to examine the virulence phenotype of the ibeR deletion mutant, a comparative study of the invasiveness of E44 (parent
strain), BR2, and the complemented BR2 was carried out. As shown in Figure 1(c),
the relative invasion rate of BR2 was significantly reduced as compared to that
of E44 and the plasmid pWKS1030 carrying the ibeR gene was able to complement the noninvasive phenotype of BR2,
suggesting that the ibeR gene contributes to the E. coli E44
invasion process.Generation
and characterization of   ibeR  deletion mutant from the meningitic strain
E44.  (a) Generation of the ibeR deletion mutant (BR2). Two DNA fragments
flanking ibeR were amplified and ligated into the suicide vector pCVD442
to construct pCBR2. The ibeR deletion mutants were generated through
gene allele exchanges and suicide vector loss. The arrowheads indicate primer
locations. (b) Verification of the ibeR deletion by PCR. The parent
strain E44 and the ibeR deletion mutant BR2 were verified though colony
PCR with primers listed in Table 2. (c) Invasion of HBMEC with the E. coli parent E44 carrying pWKS30, the ibeR mutant BR2 with pWKS30, and the complemented BR2 carrying the ibeR locus in pWKS30. E44/pWKS, BR2/Pwks,
and BR2/pWKS1030 were incubated with HBMEC monolayers and the standard invasion
assay was carried out as described in Section
2.
The results are expressed as relative
invasion%. Columns marked with  **  are significantly different (P<.01).
(a)(b)(c)


### 3.2. 2d Proteomic Analyses of Iber-Regulated SP Protein Expression in Meningitic Strain E44

To determine the role ofibeR in regulating SP gene expressionof meningitic E. coli K1, the wild type E. coli E44 and its ibeR deletion mutant BR2
were cultured in BHI broth overnight. The total proteins of each strain were
extracted from the cells as described in Section 2.  The whole cell extracts were analyzed on the
2D protein gels. Approximately 800 spots were detected on a gel image. The
experiment was performed three times with two sets of independently grown
cultures. Only spots showing the same pattern in three independent runs were
retained and quantified using the software ImageMaster 2D Platinum version 6.0.
Figures 2(a) and 2(b) showed the protein patterns of E44 and BR2, respectively.
All the upregulated spots and downregulated spots satisfying the criteria as
mentioned above were marked on both the 2D maps. They were excised and
identified by LC-MS/MS (Table 3). Eight protein spots were found to be
differentially expressed in BR2
as compared to its parent strain E44. Among them, 4 protein spots were
significantly upregulated in BR2 including elongation factor EF-Tu (TufB, spot U1),
glyceraldehyde-3-phosphate dehydrogenase A (GapA, spot U2), outer membrane protein 3a (OmpA, spot U3), and alkyl hydroperoxide
reductase (AhpC, spot U4),
while 4 protein spots were of decreased abundance in BR2, including
dihydrolipoamide dehydrogenase (LpdA,
spot D1), tryptophanase (TnaA,
spot D2), and two isoforms of outer membrane protein C (OmpC, spot D3, and D4). Figure 3(a) showed the enlargements of
each changed protein marked with black arrows and spot numbers. The relative
ratios of each downregulated protein and upregulated protein were shown in Figures
3(b) and 3(c).Table 3
Identification of differentially displayed proteins in 2D maps.
Spot numberProtein IDAccess numberMass(KD)/PI  (theriol)Mass(KD)/PI
(analytic)Peptides matchedSequence coverageFunction categorydownregulatedD1dihydrolipoamide dehydrogenase
(LpdA)gi|1579980050.9/5.7951.0/6.0524947%Central metabolism (CM): E3
component of pyruvate dehydrogenase complexD2tryptophanase (TnaA)gi|1580430553.8/5.8849.0/6.109234%Response to environmental
modifications (REMs), and CM:  initiation
of indole signalingD3outer membrane protein C (OmpC)gi|1580276840.5/4.5540.5/4.555350%REM: osmotically regulated porinD4outer membrane protein C (OmpC)gi|1580276840.5/4.5540.5/4.60720%REM: osmotically regulated porinupregulatedU1elongation
factor EF-Tu (TufB)gi|1580385243.4/5.343.5/5.305545%REM:  binding and transport of aminoacyl-tRNAU2glyceraldehyde-3-phosphate (G-3P)
dehydrogenase A(GapA)gi|1580219335.7/6.6136.0/6.209432%CM: oxidation and phosphorylation
of G-3P to 1,3-bisphosphoglycerateU3outer
membrane protein 3a (OmpA)gi|1580081637.3/5.9930.0/5.803548%REM: maintaining cell envelope integrityU4alkyl hydroperoxide reductase(AhpC)gi|1580032020.9/5.0322.0/5.155749%REM: a primary scavenger of endogenous  H2O2 at a low (10−5 M) concentration2DE maps of E. coli strains with stationary-phase cultures in BHI medium. (a) E44 (wild type strain). (b) BR2
(the ibeR deletion mutant). The upregulated proteins were circled with a
solid line and marked with U1–U4; the downregulated proteins were circled with
a broken line and marked D1–D4.
(a)(b)Comparative
analysis of the protein spots showing significant changes. (a) Enlargement of differentially expressed protein spots (indicated with
arrows) from Figure 2. (b) The relative level of down-regulated proteins. (c)
The relative level of up-regulated proteins. The protein spot intensities of
E44 were showed as black columns, and the protein spot intensities of BR2 were
showed as white columns. Columns marked with ** are significantly different (P<0.01).
(a)We classified the proteins into two main categories on the
basis of their roles in the SP growth ofE. coli cells: (a) response to
environmental modifications (including TnaA,
TufB, OmpC, OmpA, and AhpC)
and (b) central metabolism (including LpdA
and GapA). Since ibeR was hypothesized as an
SP-regulator contributing to the growth regulation and virulence of E44, its
role in the invasion process and resistance to stress conditions should be
further characterized. TnaA, a tryptophanase, degrades tryptophan, resulting in
the formation of indole, which has been proposed to act as an extracellular
signal in stationary phase cells of E. coli [28, 29]. Production of
indole, via the enzymatic activity of TnaA, is also induced during biofilm
formation [30]. TnaA, which was
controlled by RpoS in other E. coli strains [27], is one of the most
important transcriptional regulators for the gene expressions in SP cells [31].
TufB (EF-Tu) is responsible for binding and transporting the appropriate
codon-specified aminoacyl-tRNA to the aminoacyl (A) site of the ribosome [32, 33]. In addition to its function in translation elongation, elongation factor
Tu is implicated in protein folding and protection from stress like a chaperone
molecule [34].OmpC, as well as OmpF, is a porin protein present on the
outer membrane ofE. coli, responding to the osmotic challenge. OmpR, as
a regulator, activates transcription of ompF and ompC [35], and
changes the ratio of these two, so that the total level of porin proteins
remains approximately constant [36, 37]. OmpF, which produces slightly wider
pores (1.2 nm) than does OmpC, predominates at low osmotic strength, whereas
OmpC (1.1 nm) predominates at high osmotic strength [38]. In E. coli,
the expression of OmpC is repressed at low osmolarity and induced at high
osmolarity. It has been proposed that the smaller pores formed by OmpC could
reduce the diffusion of larger hydrophobic and negatively charged molecules
when bacteria encounter high osmolarity conditions as in the host compartments.
Presumably, this protein is very important for the stress resistance of E.
coli in the stationary phase. In this study, the downregulation of OmpC
resulting from the ibeR deletion might decrease resistance to high
osmolarity. In addition, it had been reported that OmpC is involved in invasion
of epithelial cells by Crohn's disease-associated E. coli strain LF82 and Shigella flexneri [39, 40], suggesting
that OmpC might be involved in E44 invasion of the host tissue barriers. OmpA
is a major protein in the outer membrane of both pathogenic and nonpathogenic
E. coli
[41]. As shown in our previous study, the ompA-deletion mutant of E44 was
significantly more sensitive than that of its parent strain to SDS, cholate,
acidic environment, and high osmolarity [41]. OmpA is downregulated upon entry
into SP by sigmaE, which plays a central role in maintaining cell envelope
integrity both under stress conditions and during normal growth [42, 43]. We
demonstrated here that OmpA was upregulated in the ibeR mutant BR2 (Figure 3), suggesting that OmpA expression is suppressed
upon entry  into SP by IbeR in a manner similar to sigmaE.Alkyl hydroperoxidase (AhpC) functions as a primary scavenger of endogenous H2O2 at a low (10−5 M) concentration [44]. All of ahpC, katG,
and katE genes are known to participate in the antioxidant defense
mechanism against H2O2-induced stress in E. coli.
It has been reported that SP-inducible RpoS regulates katE gene
expression and OxyR regulates ahpC and katG genes [45, 46]. Our
previous study has demonstrated that E. coli K1 RS218 had a nonsense
mutation in its rpoS gene, resulting in a negligible katE activity, but no obvious difference in katG [5]. In this study, the
increase in ahpC expression indicated that the ibeR deletion led to an increased oxidative stress in SP compared with the wild type
strain, suggesting that ibeR is involved in the resistance to oxidative
stress upon entry into SP.Lipoamide
dehydrogenase (LpdA), which is the same as dihydrolipoamide
dehydrogenase (DLDH), makes up the E3 component of pyruvate dehydrogenase
complex, 2-oxo glutarate dehydrogenase, and branched-chain 2-oxo acid
dehydrogenase complexes. DLDH has been identified as virulence factors
contributing to the pathogenesis of bacterial infections caused byMycobacterium tuberculosis and Streptococcus pneumoniae because it
enhances their survival within the host cells [47, 48]. As shown in Figure 3,
LpdA was downregulated in the ibeR deletion mutant BR2, suggesting that this enzyme might be involved in the
virulence of meningitic E. coli K1 via enhancing the pathogen survival
within the host. Recently, GapA in E.
coli has been identified as one of a few proteins, which harbors
functionally important thiol groups against oxidative stress [49]. As GapA is
upregulated in BR2 (Figure 3), IbeR may be involved in the negative control of gapA in SP.In summary, all
these proteins contribute to growth-related carbon source metabolism or stress
resistance. They are associated with the SP regulation. Among these proteins, TnaA is the most
important one as it produces the signal molecule indole and is regulated by
RpoS [27]. Since E44 carries a loss-of-function mutation in its rpoS gene [5], there should be alternative signaling pathway(s) to complement the
functional deficiency of RpoS in this pathogenic E. coli strain. Our proteomic analyses showed that the TnaA expression was significantly affected by IbeR, which might be functionally
equivalent to RpoS. Therefore, our
focus for further studies was placed on how TnaA is regulated by IbeR.


### 3.3. Indole Production Is Controlled by Iber Via Regulation of TnaA

To test our hypothesis that IbeR is an RpoS-like regulator,
thetnaA in-frame deletion mutant TNA44 was generated with the same gene replacement approach that
was used for the ibeR deletion mutant. TNA44 was obtained by mating E44 with SM10 λpir carrying the
recombinant suicide plasmid pCTNA2 which contains the truncated tnaA gene. The virulence phenotype of
TNA44 was examined with invasion assays. Although overall growth rates did not
differ between the mutants (BR2 and TNA44) and their parent strain E44 (Figure 4(a)),
the invasive capability of TNA44 (19%) and BR2 (35%) was significantly reduced
as compared to that of E44 (100%) (Figures 4(c)-4(d)). These data
suggest that TnaA is an important downstream regulator that is required forIbeR-modulated E. coli K1 invasion.Role of TnaA in indole
production and bacterial invasion. (a) Growth curves of E44, TNA44,  and BR2, (b) indole production (IP) of E coli strains E44, BR2, and TNA44, (c) the
invasion phenotypes of E44, TNA44, and indole(100 μM)-complemented mutant TNA44,
and (d) the invasion phenotypes of E44, BR2, and indole(100 μM)-complemented
mutant BR2. Columns marked with * are significantly different (P<.05),
** are significantly different (P<.01).
(a)(b)(c)(d)As our proteomics analysis had shown thattnaA expression was induced by IbeR in SP and the tnaA and ibeR deletion resulted in
a deficiency in indole production in SP-cultures (Figure 4(b)), we further tested
whether the TnaA product indole, as an SP extracellular signal molecule, played
a role in the process of invasion. Indole is converted to indigo (which is not
further degraded in E. coli) by several monooxygenases, thus providing
an easy method for its determination [12]. A plasmid pStyABB, carrying the gene
for styrene monooxygenase, was used to monitor the indole production through
its conversion to indigo. The plasmid was transformed into E. coli strains E44, BR2, and TNA44, and the indole production was measured at
different time points for these stains in BHI media (Figure 4(b)). The
production of indole in E44 was revealed by indigo accumulation. By contrast,
the indole production was almost abolished in the tnaA deletion mutant
and severely reduced in the ibeR deletion mutant (Figure 4(b)). These
results demonstrated that the indole production was controlled by ibeR through tnaA. To examine whether indole could compliment the noninvasive
phenotype of tnaA and ibeR deletion mutants, indole was supplied in the BHI medium at 100 μM to the TNA44
and BR2 SP cultures. The
result showed that indole was able to significantly
enhance the relative invasion rate of TNA44 (19% to 69%) and BR2 (35% to 65%) as compared to
that of E44, suggesting that indole could partly compliment the noninvasive phenotype of TNA44
(Figure 4(c)) and BR2 (Figure 4(d)). Lacour and Landini have shown that the rpoS gene in E. coli K12 strain
MG1655 controls the production of indole, which acts as a signal molecule in SP
cells, via regulation of TnaA, the indole-producing enzyme [27]. As TnaA is
regulated by IbeR in E44, it is most likely that IbeR is a novel regulator to
complement the functional deficiency of RpoS in E44.


### 3.4. The Role ofibeR in Stress Conditions

It has been reported that RpoS is able to positively and
negatively control expression of a large set of genes when bacteria enter into
the SP [46, 50, 51]. During such
transition, bacteria undergo physiological changes that allow their SP
organisms to survive better in such insults as heat, high-osmotic environment,
starvation, UV radiation, H2O2,  and acid than their
exponential counterpart [50, 52]. The loss of RpoS resulted in the decrease of
stress resistance and cell survival in the SP [5, 53]. Although our study
showed that the loss of ibeR did not affect the growth rate in BHI
medium, the survival rates of the ibeR deletion mutant BR2 in the SP
significantly decreased as compared to that of the wild type strain E44 even
without any stress treatment (Figure 5(a)). Our proteomics analysis also
revealed that the most significant proteomic changes in the ibeR deletion mutant were related to bacterial response to environmental
modifications. For example, AhpC, as a primary scavenger of endogenous H2O2,
was upregulated in the ibeR  deletion mutant, implying that the loss of ibeR resulted in the decreased survival rates of bacterial cells under an oxidative
stress in the SP. OmpC, as a porin protein, was downregulated in BR2, perhaps
resulting in the decreased resistance to osmolarity stress. These results
suggested that ibeR plays a regulatory role in response to stress
conditions in E44 that carries a nonsense mutation in rpoS. To examine
the function of ibeR in response to stress environments, we performed
several survival assays under different stress conditions, including heat shock
(54°C for 3 minutes), alkali endurance (Tris, pH = 10 for 30 minutes), acid
endurance (acetic acid, pH = 2.8 for 20 minutes), high osmolarity challenge (2.5 M NaCl for 1 hour), and oxidative stress (10 μM H2O2 for
30 minutes). In all the survival experiments, the wild type strain E44 showed
higher survival rates than the ibeR deletion mutant, indicating that the ibeR gene is required for all these
stress resistances (Figure 5(b)). Especially in the heat shock assay, the loss
of ibeR resulted in over 95% cells death, indicating that ibeR played a vital role in temperature sensitivity in this strain. In the other
stress treatments, the ibeR deletion also significantly reduced the
survival rates (more than 60% cell death) of BR2 as compared to that of the
wild type strain E44, suggesting that IbeR had a global regulatory role in the
resistance to acid, alkali, high osmolarity and oxidative stress. In the
survival assays for the E. coli control strain MG1655, the rpoS deletion mutant RH90 also decreased the survival levels in these five stress
conditions, showing the similar patterns like ibeR in response to stress
environments (data not shown). Combining the proteomics analysis and the stress
survival studies, we conclude that IbeR is an RpoS-like regulator to control
gene expression of proteins that are critical for stress-resistance and cell
survival in the SP in E44, which carries a loss-of-function mutation in the rpoS gene.The relative survival rate
of the BR2 mutant comparing with E44.  (a) Both strains in stationary phase without
any stress treatment, (b) both strains were tested with different environmental
stress including heat shock (54°C for 3 minutes), alkali (Tris pH = 10.0 for 30 minutes),
acid endurance (acetic acid, pH = 2.8 for 20 minutes), high osmolarity challenge
(2.4 M NaCl for 1 hour), and oxidative stress (10 μM  H2O2 for 30 minutes). Gray columns: E44; white columns: BR2. Columns marked with **
are significantly different (P<.01).
(a)(b)


## 3.1. TheibeR Regulatory Gene Is Required for Invasion of Human BMEC by Meningitic E.   coli K1 Strain E44

The geneibeR in E. coli K1 E44 is predicted as
the only regulatory protein present in the ibeRAT operon in GimA by
bioinformatics approaches [16]. To determine the role of ibeR gene in
the growth-phase-dependent invasion of BMEC by meningitic E. coli K1, an isogenic in-frame deletion mutant of ibeR was
made by chromosomal gene replacement with the recombinant suicide plasmid pCBR2
carrying a 2.2 kb DNA fragment with ibeR internal deletion (Figure 1(a)).
The 2.2 kb DNA fragment was generated by ligation of two PCR amplicons (1.2 and
1.0 kbs) flanking the
1.8 kb ibeR coding region. The ibeR deletion mutant was obtained
by mating E44 with SM10 λpir carrying pCBR2. The mutant colony morphology on LB
agar plates and growth rate in LB broth were the same as the parent strain E44.
The deletion of ibeR was confirmed by colony PCR and DNA sequencing
(Figure 1(b)). In order to examine the virulence phenotype of the ibeR deletion mutant, a comparative study of the invasiveness of E44 (parent
strain), BR2, and the complemented BR2 was carried out. As shown in Figure 1(c),
the relative invasion rate of BR2 was significantly reduced as compared to that
of E44 and the plasmid pWKS1030 carrying the ibeR gene was able to complement the noninvasive phenotype of BR2,
suggesting that the ibeR gene contributes to the E. coli E44
invasion process.Generation
and characterization of   ibeR  deletion mutant from the meningitic strain
E44.  (a) Generation of the ibeR deletion mutant (BR2). Two DNA fragments
flanking ibeR were amplified and ligated into the suicide vector pCVD442
to construct pCBR2. The ibeR deletion mutants were generated through
gene allele exchanges and suicide vector loss. The arrowheads indicate primer
locations. (b) Verification of the ibeR deletion by PCR. The parent
strain E44 and the ibeR deletion mutant BR2 were verified though colony
PCR with primers listed in Table 2. (c) Invasion of HBMEC with the E. coli parent E44 carrying pWKS30, the ibeR mutant BR2 with pWKS30, and the complemented BR2 carrying the ibeR locus in pWKS30. E44/pWKS, BR2/Pwks,
and BR2/pWKS1030 were incubated with HBMEC monolayers and the standard invasion
assay was carried out as described in Section
2.
The results are expressed as relative
invasion%. Columns marked with  **  are significantly different (P<.01).
(a)(b)(c)


## 3.2. 2d Proteomic Analyses of Iber-Regulated SP Protein Expression in Meningitic Strain E44

To determine the role ofibeR in regulating SP gene expressionof meningitic E. coli K1, the wild type E. coli E44 and its ibeR deletion mutant BR2
were cultured in BHI broth overnight. The total proteins of each strain were
extracted from the cells as described in Section 2.  The whole cell extracts were analyzed on the
2D protein gels. Approximately 800 spots were detected on a gel image. The
experiment was performed three times with two sets of independently grown
cultures. Only spots showing the same pattern in three independent runs were
retained and quantified using the software ImageMaster 2D Platinum version 6.0.
Figures 2(a) and 2(b) showed the protein patterns of E44 and BR2, respectively.
All the upregulated spots and downregulated spots satisfying the criteria as
mentioned above were marked on both the 2D maps. They were excised and
identified by LC-MS/MS (Table 3). Eight protein spots were found to be
differentially expressed in BR2
as compared to its parent strain E44. Among them, 4 protein spots were
significantly upregulated in BR2 including elongation factor EF-Tu (TufB, spot U1),
glyceraldehyde-3-phosphate dehydrogenase A (GapA, spot U2), outer membrane protein 3a (OmpA, spot U3), and alkyl hydroperoxide
reductase (AhpC, spot U4),
while 4 protein spots were of decreased abundance in BR2, including
dihydrolipoamide dehydrogenase (LpdA,
spot D1), tryptophanase (TnaA,
spot D2), and two isoforms of outer membrane protein C (OmpC, spot D3, and D4). Figure 3(a) showed the enlargements of
each changed protein marked with black arrows and spot numbers. The relative
ratios of each downregulated protein and upregulated protein were shown in Figures
3(b) and 3(c).Table 3
Identification of differentially displayed proteins in 2D maps.
Spot numberProtein IDAccess numberMass(KD)/PI  (theriol)Mass(KD)/PI
(analytic)Peptides matchedSequence coverageFunction categorydownregulatedD1dihydrolipoamide dehydrogenase
(LpdA)gi|1579980050.9/5.7951.0/6.0524947%Central metabolism (CM): E3
component of pyruvate dehydrogenase complexD2tryptophanase (TnaA)gi|1580430553.8/5.8849.0/6.109234%Response to environmental
modifications (REMs), and CM:  initiation
of indole signalingD3outer membrane protein C (OmpC)gi|1580276840.5/4.5540.5/4.555350%REM: osmotically regulated porinD4outer membrane protein C (OmpC)gi|1580276840.5/4.5540.5/4.60720%REM: osmotically regulated porinupregulatedU1elongation
factor EF-Tu (TufB)gi|1580385243.4/5.343.5/5.305545%REM:  binding and transport of aminoacyl-tRNAU2glyceraldehyde-3-phosphate (G-3P)
dehydrogenase A(GapA)gi|1580219335.7/6.6136.0/6.209432%CM: oxidation and phosphorylation
of G-3P to 1,3-bisphosphoglycerateU3outer
membrane protein 3a (OmpA)gi|1580081637.3/5.9930.0/5.803548%REM: maintaining cell envelope integrityU4alkyl hydroperoxide reductase(AhpC)gi|1580032020.9/5.0322.0/5.155749%REM: a primary scavenger of endogenous  H2O2 at a low (10−5 M) concentration2DE maps of E. coli strains with stationary-phase cultures in BHI medium. (a) E44 (wild type strain). (b) BR2
(the ibeR deletion mutant). The upregulated proteins were circled with a
solid line and marked with U1–U4; the downregulated proteins were circled with
a broken line and marked D1–D4.
(a)(b)Comparative
analysis of the protein spots showing significant changes. (a) Enlargement of differentially expressed protein spots (indicated with
arrows) from Figure 2. (b) The relative level of down-regulated proteins. (c)
The relative level of up-regulated proteins. The protein spot intensities of
E44 were showed as black columns, and the protein spot intensities of BR2 were
showed as white columns. Columns marked with ** are significantly different (P<0.01).
(a)We classified the proteins into two main categories on the
basis of their roles in the SP growth ofE. coli cells: (a) response to
environmental modifications (including TnaA,
TufB, OmpC, OmpA, and AhpC)
and (b) central metabolism (including LpdA
and GapA). Since ibeR was hypothesized as an
SP-regulator contributing to the growth regulation and virulence of E44, its
role in the invasion process and resistance to stress conditions should be
further characterized. TnaA, a tryptophanase, degrades tryptophan, resulting in
the formation of indole, which has been proposed to act as an extracellular
signal in stationary phase cells of E. coli [28, 29]. Production of
indole, via the enzymatic activity of TnaA, is also induced during biofilm
formation [30]. TnaA, which was
controlled by RpoS in other E. coli strains [27], is one of the most
important transcriptional regulators for the gene expressions in SP cells [31].
TufB (EF-Tu) is responsible for binding and transporting the appropriate
codon-specified aminoacyl-tRNA to the aminoacyl (A) site of the ribosome [32, 33]. In addition to its function in translation elongation, elongation factor
Tu is implicated in protein folding and protection from stress like a chaperone
molecule [34].OmpC, as well as OmpF, is a porin protein present on the
outer membrane ofE. coli, responding to the osmotic challenge. OmpR, as
a regulator, activates transcription of ompF and ompC [35], and
changes the ratio of these two, so that the total level of porin proteins
remains approximately constant [36, 37]. OmpF, which produces slightly wider
pores (1.2 nm) than does OmpC, predominates at low osmotic strength, whereas
OmpC (1.1 nm) predominates at high osmotic strength [38]. In E. coli,
the expression of OmpC is repressed at low osmolarity and induced at high
osmolarity. It has been proposed that the smaller pores formed by OmpC could
reduce the diffusion of larger hydrophobic and negatively charged molecules
when bacteria encounter high osmolarity conditions as in the host compartments.
Presumably, this protein is very important for the stress resistance of E.
coli in the stationary phase. In this study, the downregulation of OmpC
resulting from the ibeR deletion might decrease resistance to high
osmolarity. In addition, it had been reported that OmpC is involved in invasion
of epithelial cells by Crohn's disease-associated E. coli strain LF82 and Shigella flexneri [39, 40], suggesting
that OmpC might be involved in E44 invasion of the host tissue barriers. OmpA
is a major protein in the outer membrane of both pathogenic and nonpathogenic
E. coli
[41]. As shown in our previous study, the ompA-deletion mutant of E44 was
significantly more sensitive than that of its parent strain to SDS, cholate,
acidic environment, and high osmolarity [41]. OmpA is downregulated upon entry
into SP by sigmaE, which plays a central role in maintaining cell envelope
integrity both under stress conditions and during normal growth [42, 43]. We
demonstrated here that OmpA was upregulated in the ibeR mutant BR2 (Figure 3), suggesting that OmpA expression is suppressed
upon entry  into SP by IbeR in a manner similar to sigmaE.Alkyl hydroperoxidase (AhpC) functions as a primary scavenger of endogenous H2O2 at a low (10−5 M) concentration [44]. All of ahpC, katG,
and katE genes are known to participate in the antioxidant defense
mechanism against H2O2-induced stress in E. coli.
It has been reported that SP-inducible RpoS regulates katE gene
expression and OxyR regulates ahpC and katG genes [45, 46]. Our
previous study has demonstrated that E. coli K1 RS218 had a nonsense
mutation in its rpoS gene, resulting in a negligible katE activity, but no obvious difference in katG [5]. In this study, the
increase in ahpC expression indicated that the ibeR deletion led to an increased oxidative stress in SP compared with the wild type
strain, suggesting that ibeR is involved in the resistance to oxidative
stress upon entry into SP.Lipoamide
dehydrogenase (LpdA), which is the same as dihydrolipoamide
dehydrogenase (DLDH), makes up the E3 component of pyruvate dehydrogenase
complex, 2-oxo glutarate dehydrogenase, and branched-chain 2-oxo acid
dehydrogenase complexes. DLDH has been identified as virulence factors
contributing to the pathogenesis of bacterial infections caused byMycobacterium tuberculosis and Streptococcus pneumoniae because it
enhances their survival within the host cells [47, 48]. As shown in Figure 3,
LpdA was downregulated in the ibeR deletion mutant BR2, suggesting that this enzyme might be involved in the
virulence of meningitic E. coli K1 via enhancing the pathogen survival
within the host. Recently, GapA in E.
coli has been identified as one of a few proteins, which harbors
functionally important thiol groups against oxidative stress [49]. As GapA is
upregulated in BR2 (Figure 3), IbeR may be involved in the negative control of gapA in SP.In summary, all
these proteins contribute to growth-related carbon source metabolism or stress
resistance. They are associated with the SP regulation. Among these proteins, TnaA is the most
important one as it produces the signal molecule indole and is regulated by
RpoS [27]. Since E44 carries a loss-of-function mutation in its rpoS gene [5], there should be alternative signaling pathway(s) to complement the
functional deficiency of RpoS in this pathogenic E. coli strain. Our proteomic analyses showed that the TnaA expression was significantly affected by IbeR, which might be functionally
equivalent to RpoS. Therefore, our
focus for further studies was placed on how TnaA is regulated by IbeR.


## 3.3. Indole Production Is Controlled by Iber Via Regulation of TnaA

To test our hypothesis that IbeR is an RpoS-like regulator,
thetnaA in-frame deletion mutant TNA44 was generated with the same gene replacement approach that
was used for the ibeR deletion mutant. TNA44 was obtained by mating E44 with SM10 λpir carrying the
recombinant suicide plasmid pCTNA2 which contains the truncated tnaA gene. The virulence phenotype of
TNA44 was examined with invasion assays. Although overall growth rates did not
differ between the mutants (BR2 and TNA44) and their parent strain E44 (Figure 4(a)),
the invasive capability of TNA44 (19%) and BR2 (35%) was significantly reduced
as compared to that of E44 (100%) (Figures 4(c)-4(d)). These data
suggest that TnaA is an important downstream regulator that is required forIbeR-modulated E. coli K1 invasion.Role of TnaA in indole
production and bacterial invasion. (a) Growth curves of E44, TNA44,  and BR2, (b) indole production (IP) of E coli strains E44, BR2, and TNA44, (c) the
invasion phenotypes of E44, TNA44, and indole(100 μM)-complemented mutant TNA44,
and (d) the invasion phenotypes of E44, BR2, and indole(100 μM)-complemented
mutant BR2. Columns marked with * are significantly different (P<.05),
** are significantly different (P<.01).
(a)(b)(c)(d)As our proteomics analysis had shown thattnaA expression was induced by IbeR in SP and the tnaA and ibeR deletion resulted in
a deficiency in indole production in SP-cultures (Figure 4(b)), we further tested
whether the TnaA product indole, as an SP extracellular signal molecule, played
a role in the process of invasion. Indole is converted to indigo (which is not
further degraded in E. coli) by several monooxygenases, thus providing
an easy method for its determination [12]. A plasmid pStyABB, carrying the gene
for styrene monooxygenase, was used to monitor the indole production through
its conversion to indigo. The plasmid was transformed into E. coli strains E44, BR2, and TNA44, and the indole production was measured at
different time points for these stains in BHI media (Figure 4(b)). The
production of indole in E44 was revealed by indigo accumulation. By contrast,
the indole production was almost abolished in the tnaA deletion mutant
and severely reduced in the ibeR deletion mutant (Figure 4(b)). These
results demonstrated that the indole production was controlled by ibeR through tnaA. To examine whether indole could compliment the noninvasive
phenotype of tnaA and ibeR deletion mutants, indole was supplied in the BHI medium at 100 μM to the TNA44
and BR2 SP cultures. The
result showed that indole was able to significantly
enhance the relative invasion rate of TNA44 (19% to 69%) and BR2 (35% to 65%) as compared to
that of E44, suggesting that indole could partly compliment the noninvasive phenotype of TNA44
(Figure 4(c)) and BR2 (Figure 4(d)). Lacour and Landini have shown that the rpoS gene in E. coli K12 strain
MG1655 controls the production of indole, which acts as a signal molecule in SP
cells, via regulation of TnaA, the indole-producing enzyme [27]. As TnaA is
regulated by IbeR in E44, it is most likely that IbeR is a novel regulator to
complement the functional deficiency of RpoS in E44.


## 3.4. The Role ofibeR in Stress Conditions

It has been reported that RpoS is able to positively and
negatively control expression of a large set of genes when bacteria enter into
the SP [46, 50, 51]. During such
transition, bacteria undergo physiological changes that allow their SP
organisms to survive better in such insults as heat, high-osmotic environment,
starvation, UV radiation, H2O2,  and acid than their
exponential counterpart [50, 52]. The loss of RpoS resulted in the decrease of
stress resistance and cell survival in the SP [5, 53]. Although our study
showed that the loss of ibeR did not affect the growth rate in BHI
medium, the survival rates of the ibeR deletion mutant BR2 in the SP
significantly decreased as compared to that of the wild type strain E44 even
without any stress treatment (Figure 5(a)). Our proteomics analysis also
revealed that the most significant proteomic changes in the ibeR deletion mutant were related to bacterial response to environmental
modifications. For example, AhpC, as a primary scavenger of endogenous H2O2,
was upregulated in the ibeR  deletion mutant, implying that the loss of ibeR resulted in the decreased survival rates of bacterial cells under an oxidative
stress in the SP. OmpC, as a porin protein, was downregulated in BR2, perhaps
resulting in the decreased resistance to osmolarity stress. These results
suggested that ibeR plays a regulatory role in response to stress
conditions in E44 that carries a nonsense mutation in rpoS. To examine
the function of ibeR in response to stress environments, we performed
several survival assays under different stress conditions, including heat shock
(54°C for 3 minutes), alkali endurance (Tris, pH = 10 for 30 minutes), acid
endurance (acetic acid, pH = 2.8 for 20 minutes), high osmolarity challenge (2.5 M NaCl for 1 hour), and oxidative stress (10 μM H2O2 for
30 minutes). In all the survival experiments, the wild type strain E44 showed
higher survival rates than the ibeR deletion mutant, indicating that the ibeR gene is required for all these
stress resistances (Figure 5(b)). Especially in the heat shock assay, the loss
of ibeR resulted in over 95% cells death, indicating that ibeR played a vital role in temperature sensitivity in this strain. In the other
stress treatments, the ibeR deletion also significantly reduced the
survival rates (more than 60% cell death) of BR2 as compared to that of the
wild type strain E44, suggesting that IbeR had a global regulatory role in the
resistance to acid, alkali, high osmolarity and oxidative stress. In the
survival assays for the E. coli control strain MG1655, the rpoS deletion mutant RH90 also decreased the survival levels in these five stress
conditions, showing the similar patterns like ibeR in response to stress
environments (data not shown). Combining the proteomics analysis and the stress
survival studies, we conclude that IbeR is an RpoS-like regulator to control
gene expression of proteins that are critical for stress-resistance and cell
survival in the SP in E44, which carries a loss-of-function mutation in the rpoS gene.The relative survival rate
of the BR2 mutant comparing with E44.  (a) Both strains in stationary phase without
any stress treatment, (b) both strains were tested with different environmental
stress including heat shock (54°C for 3 minutes), alkali (Tris pH = 10.0 for 30 minutes),
acid endurance (acetic acid, pH = 2.8 for 20 minutes), high osmolarity challenge
(2.4 M NaCl for 1 hour), and oxidative stress (10 μM  H2O2 for 30 minutes). Gray columns: E44; white columns: BR2. Columns marked with **
are significantly different (P<.01).
(a)(b)


## 4. Concluding Remarks

Currently, mostE. coli meningitis studies are done with SP cultures in which the pathogen invasion of
human BMEC is significantly greater than the log phase cultures. In most
strains of E. coli, RpoS plays a
central role in regulating the SP regulatory genes for protecting cells against
starvation and stress damage. RpoS, the major SP regulator, has also been shown
to regulate the expression of microbial virulence genes in various bacteria
including E. coli K1 (O157:H7), Salmonella
typhimurium, Shigella flexneri, Yersinia enterocolitica, Vibrio
cholera, and Borrelia burgdorfer,  [5, 53, 54]. Surprisingly, however, RpoS was found to be inactive in meningitic
strain E44 [5]. The current proteomic studies may provide an answer to the
long-standing question regarding the SP gene regulation in E44. Combining the
proteomics analysis, virulence determination, and the stress survival studies
of the ibeR mutant BR2, we have
demonstrated for the first time that IbeR serves as an RpoS-like regulator to
control gene expression that is critical for stress-resistance and cell
survival in the SP of E44.IbeR is not a structural homologue of  RpoS as IbeR
and RpoS do not share any significant sequence homology. RpoS (also known asσ38,
σs, or KatF) is a global regulator in E. coli, which is the second principal σ subunit after the major σ70 factor [5]. In E44, however, IbeR appears not to be a master regulator on the
basis of its genomic prevalence and functional spectrum. The prevalence of the
GimAlocus carrying ibeR is highly dependent on the origin
of the strain and on the subgroup it belongs to (A, B1, B2, and D) (4). In all
the studies, where the presence of this locushas been analyzed, GimA was found to be restricted to the B2
subgroup, a subgroup that includes strains with the highest virulence in mice
and the highest level of virulence determinants (4). Our proteomic studies showed that a limited number
of genes were regulated by IbeR, suggesting that IbeR is a regulator with a
narrow functional spectrum. In other E.
coli strains, either pathogenic or probiotic strains, functional
heterogeneity of RpoS in stress tolerance was widely observed [5, 53, 55].
Those studies have shown that some E.
coli strains can maintain their stress tolerance capability or
significantly modulate their stress resistance phenotype independent of their rpoS genotypes. Such adaptation processes
compromising the RpoS-dependent stress responses may have significant impact on
bacterial survival in environments, as well as in the host's stomach and
intestine [53, 55]. IbeR, a regulator in the GimA regulon, may contribute to
bacterial virulence adaptation process in E44 to complement the functional
deficiency of RpoS.Another significant finding of our proteomic studies is that
IbeR in meningitic strain E44 is able to upregulate TnaA, which is controlled
by RpoS in otherE. coli strains
[56]. The virulence determination of the tnaA mutant showed that TnaA and its product indole were required for E44 invasion
of human BMEC. The generation of indole, via the tryptophanase activity of
TnaA, was also observed during the formation of biofilm in E. coli and other bacteria [28, 56]. In addition to the initiation
of indole-mediated signaling, TnaA (tryptophanase) is able to catabolize
tryptophan, cysteine, and serine to pyruvate [29, 56]. The Three proteins
significantly upregulated by IbeR are TnaA, LpdA, and OmpC, all of which are
directly or indirectly involved in pyruvate metabolism. LpdA (dihydrolipoamide
dehydrogenase) is the E3 component of pyruvate dehydrogenase complex. OmpC, an
osmotically regulated porin, may facilitate nutrient uptake [56]. On the other
hand, the three operons (ptnIPKC, cglDTEC, and gcxKRCI) in the GimA regulon may also directly or indirectly
contribute to pyruvate metabolism by converting dihydroxyacetone, glycerol, and
glycerate to pyruvate [16]. It has been shown that the capability to catabolize
carbon source is an important parameter in the ability to persist and compete
in stationary phase [29]. This raises the possibility that signaling by indole,
which is regulated by IbeR via TnaA, may play a critical role in the pathways that
prepare the pathogens for a nutrient-poor environment (e.g., CNS) when the
carbon source becomes limited for energy production.



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*Source: 520283-2009-03-12.xml*