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 .
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 .
a nonsense mutation in the sp regulatory gene rpos was identified in e. coli k1 strains e44 and ihe3034 . 

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 .
several virulence 
 factors , including ibe ( termed after   invasion of   brain endothelial 
 cells ) proteins [ 6 , 7 ] , ompa   , yiijp , fimh , asla , and traj 
 , 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 .
recently , vimentin has 
 been identified as an ibea - binding protein on the surface of human bmec . using the ibea gene 
 as a probe
, we have identified a 20.3 kb genomic locus as a genetic island of meningitic e. coli containing ibea ( gima ) .
this 
 locus is situated between yjid and yjie , adjacent to the fim operon , and 
 absent in nonpathogenic e. coli k12 
 strains .
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 .
this is 
 consistent with the observations by others that carbon source modulates 
 expression of virulence factors in several pathogenic bacteria .
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 .
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 

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
our studies suggested that the iber gene was involved in 
 regulating sp gene expression related to stress resistance and pathogenesis .
the bacterial strain and plasmid vectors and their relevant 
 characteristics are shown in table 1 .
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 ] .
sm10 ( pir ) , dh5   ( pir ) , and pcvd442 were used for making 
 isogenic deletion mutants of iber and tnaa [ 6 , 10 , 11 ] .
e. 
 coli k12 strain mc4100   and its rpos 
					 insertion mutation tn10 mutant rh90 
 were used as positive and negative controls for rpos , respectively .
plasmid 
 pstyabb , which carries the gene for monooxygenase , was used for indole assay 
 .
e. coli strains were grown at 37c 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 70c in lb plus 20% 
 glycerol . when it was necessary , the medium was supplemented with ampicillin 
 ( 100 g / ml ) and rifampin ( 100 g / ml ) for the positive selection of plasmids or 
 bacterial strains ( table 1 ) . all genetic manipulations were done by using standard 
 methods , as described elsewhere .
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 ] . to determine the role of the iber gene in the 
 growth - phase - dependent e. coli k1 invasion of bmec , an isogenic deletion 
 mutant of iber
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 , and 
 the resulting recombinant plasmid was named pcbr2 .
the mutants named br2 were 
 obtained by mating e44 with sm10 pir that carries pcbr2 as described previously 
 .
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 94c for 1 minute , 58c for 1 minute , and 70c for 1.5 minutes . for the tnaa in - frame 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 ) .
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 ( 50 g / ml ) , and streptomycin ( 100 g / ml ) at 37c in 5% co2 . 
 
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 g / ml ) for 1 hour .
results were 
 expressed as percent invasion ( 100      ( number of intracellular bacteria 
 recovered)/(number of bacteria inoculated ) ) .
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 = 2.53.0 ) by 
 centrifugation at 6000      g for 10 minutes .
denaturing protein extraction ( phenol 
 extraction procedure ) was performed according to saravanan and rose .
the lyophilized pellets were 
 dissolved in rehydration buffer ( 7 m urea , 2 m thiourea , 4% chaps , 0.2% ph 310 biolytes , 1% 
 dtt ; 1 mg dry pellets for 200 l 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 l each ) were loaded on 11 cm immobilized ph 
 gradient ( ipg ) strips ( ph 47 ) . 

rehydration / loading was done passively ( no voltage ) for 1 hour , followed 
 immediately by 14 hours of active rehydration ( 50 v ) at 20c .
isoelectric 
 focusing of the ipg strips was performed at 20c using a 50 a   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 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 .
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 56c 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 l of 0.1 g/l modified 
 trypsin ( promega , wiss , usa , 
 sequencing grade ) in 25 mm nh4hco3 was added over the gel 
 spots and incubated for 30 minutes .
about 710 l of 25 mm nh4hco3 was then added to cover the gel spots and incubated at 37c 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 .
the sample was 
 resuspended in 10 l 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 190c .
the column 
 equilibrated for 5 minutes at 1.5 l / 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 ) .
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 . 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 g / ml ampicillin overnight with shaking .
then the bacteria were collected by 
 centrifugation and resuspended to od600 = 0.2 in bhi medium supplied 
 with 100 g / ml ampicillin , and incubated at 37c without shaking . to determine 
 the indigo formation at different time points ,
the samples were read at 600 nm to determine the indigo concentration 
 by comparison to a standard curve .
bacteria were grown in bhi broth at 37c 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 10 cells / ml in pbs for the following assays . for heat shock , 100 l of bacteria 

was heated at 54c 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 37c 
 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 37c 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 37c for 1 hour . for oxidative stress ,
bacteria were harvested and 
 resuspended in an equal volume of pbs containing 10 m h2o2 incubated at 37c   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 .
the gene iber in e. coli k1 e44 is predicted as 
 the only regulatory protein present in the iberat operon in gima by 
 bioinformatics approaches . 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 pir 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 . to determine the role of iber 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 
 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 ) .
we classified the proteins into two main categories on the 
 basis of their roles in the sp growth of e. 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 .
tnaa , which was 
 controlled by rpos in other e. coli strains , is one of the most 
 important transcriptional regulators for the gene expressions in sp cells . 

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 .
ompc , as well as ompf , is a porin protein present on the 
 outer membrane of e. coli , responding to the osmotic challenge .
ompr , as 
 a regulator , activates transcription of ompf and ompc , 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 . in e. coli , 
 the expression of ompc
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 
					  .
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 .
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 m ) concentration .
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 . 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 by mycobacterium 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 .
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 . since e44 carries a loss - of - function mutation in its rpos gene , 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 . to test our hypothesis that iber is an rpos - like regulator , 
 the tnaa 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 pir carrying the 
 recombinant suicide plasmid pctna2 which contains the truncated tnaa gene .
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 .
as our proteomics analysis had shown that tnaa 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 .
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 ) ) .
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 m 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 .
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 .
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 
 ( 54c 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 m 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 .
currently , most e. 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 .
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 as  , 
  , or katf ) is a global regulator in e. coli , which is the second principal  subunit after the major  factor . 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 other e. coli strains 
 .
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 .
ompc , an 
 osmotically regulated porin , may facilitate nutrient uptake . 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 .
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 .
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 .
 
 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 . 
1. Introduction
2. Materials and Methods
3. Results and Discussions
4. Concluding Remarks