In E. coli, all the described genes inducible by paraquat are a part of the SoxRS regulon [167-169]. The expression of yjcC is RpoS-dependent in E. coli [170].
Consistent with this finding, the yjcC expression in K. pneumoniae is affected by RpoS and by SoxRS at the transcriptional level. No SoxRS or RpoS binding box appears within the putative promoter sequence, suggesting the possibility of an indirect control. In E. coli, the FNR regulator controls the transition between aerobic
30
and anaerobic growth at the transcriptional level [171]. The conserved fnr binding box present in the upstream non-coding region of yjcC implies an FNR dependent control of YjcC expression. Thus, the yjcC regulation by FNR likely occurs in poorly oxygenated environments.
The K. pneumoniae NTUH K2044 genome contains 27 genes encoding GGDEF-, EAL-, and GGDEF-EAL-domain proteins of potential DGC and PDE enzyme activity [172]. The yjcC encoding gene is also identified as member of the protein family. The family regulation specificity is determined by the sensory domain of the DGC or PDE proteins. Figure 2.2 (A) shows that the PDE expression plasmids pfimK and pmrkJ, which contain the respective coding region with putative promoter, failed to complement yjcC deletion. This may be due to fimK and mrkJ genes are not induced in comparison to yjcC in the presence of paraquat. There is also the possibility that the N-terminal peptide of approximately 300 aa of YjcC plays a role in the oxidative stress response besides signal sensing. Various sensory domains can bind to small molecular signals, and through this connection, modulate the levels of c-di-GMP [173,174]. Although the signal for the CSS-motif remains unknown, we speculate that YjcC-mediated signaling sensing may occur in the periplasmic space because of the signal peptide and the transmembrane domain at the N-terminal region.
The purified recombinant EAL domain of YjcC exhibits PDE activity. However, the
31
amount of c-di-GMP in CG43S3ΔyjcC[pJR3] is approximately the same as that in CG43S3ΔyjcC[pRK415] and CG43S3ΔyjcC[pJR2], suggesting that the EAL domain
has extremely low levels of PDE activity. The N terminal region of YjcC likely requires receiving some signal from outside, and the interaction of the sensory domain with signaling molecules activates the PDE activity. This is supported by Fig. 2.2 (D) showing that the deletion of yjcC gene from CG43S3 raised the c-di-GMP levels and the influence was much more apparent after the bacteria exposure to 30 µM of paraquat.
The deletion of yjcC decreases the CPS production and the virulence attenuation, indicating that YjcC is required to avoid the damage of oxidative stress. In bacteria, superoxide dismutase and catalase are common antioxidant enzymes that scavenge ROS from oxidative stress. The redox proteins (including GrxA and YbbN) required for maintaining redox status in bacteria are also protect bacteria from oxidative stress [175,176]. The deletion of yjcC has no apparent effect on the SOD and catalase activity, but appears to increase the transcription of grxA and ybbN. This suggests that YjcC is involved in regulating the redox levels in bacteria after oxidative stress. The chaperone protein ClpB stabilizes protein and suppresses the protein aggregation induced by heat or other stresses [165,177]. The phage shock proteins, PspB and PspC, act as positive regulators to transduce stress signal(s) to PspA through protein–protein
32
interaction, maintaining the proton-motive force under extracytoplasmic stress conditions [166]. Transcriptome analysis shows that an increase in YjcC induces the expression of several heat shock proteins and chaperones, suggesting that YjcC is involved in regulating anti-stress responses. The fumarase gene fumB, which is expressed under anaerobic cell growth conditions, is regulated by Fnr and ArcA [178,179]. The result of yjcC expression reduced the fumB transcripts implies that YjcC is a component of the Fnr and ArcA regulatory pathway.
In K. pneumoniae, the second messenger c-di-GMP activates type 3 fimbriae expression through MrkHI activation [146]. However, the increased synthesis of mrkH and mrkI transcripts by the overexpression of yjcC remains unclear. We have
found that mrkHI expression is barely detected under LB culture (unpublished observation). Under oxidative stress pressure, the N-terminal region of YjcC may turn on the expression of MrkHI. Thus, the N-terminal region likely plays a determinant role in YjcC-dependent regulation. E. coli YdeH has c-di-GMP cyclase activity [180].
The transcriptome analysis as shown in Table S3 revealed that the transcript levels of mrkA, mrkH, and mrkI in CG43[pRK415-ydeH], with c-di-GMP level of 23.1 fmole/
mg-1 (data not shown), significantly increased compared to those of CG43S3[pJR1].
This also indicates that mrkHI expression is c-di-GMP level-dependent and the N-terminal part of YjcC plays a positive regulatory role in the expression of mrkHI.
33
Overall, these results indicate that the YjcC-mediated regulatory system is considerably more complex than expected. During infection, the transition from aerobic to microaerobic conditions or the transition from a microaerobic to oxidative stress environment, YjcC may be activated through sensory regulatory systems on the N-terminal region. Thereafter, YjcC modulates the levels of c-di-GMP to affect the expression of the downstream regulatory pathways. In conclusion, YjcC regulates the oxidative stress response, mouse virulence, CPS synthesis, biofilm formation, and type 3 fimbriae expression. This most likely occurs through the adjustment of c-di-GMP levels after receiving outside signals.
34
The tested bacterial strains CG43S3, CG43S3ΔyjcC,CG43S3ΔyjcC[pJR1] and CG43S3ΔyjcC[pJR2] were cultured in LB medium at 37°C for overnight.
Five mice of a group were injected intraperitoneally with bacteria resuspended in 0.2 ml of saline in 10-fold steps graded doses. The LD50, based on the number of survivors after one week, were calculated by the method of Reed and Muench [215]and expressed as CFU.
Table 2.1. YjcC effect on the mouse virulence.
Strain LD50
CG43S3 1x104
CG43S3ΔyjcC 7.8 x105
CG43S3ΔyjcC [pJR1] 3.3 x104
CG43S3ΔyjcC [pJR2] 1.1 x106
35
Table 2.2 Significantly upregulated genes by yjcC overexpression
Primosomal replication protein N priB 2.1 KP1_0469
DNA binding protein, nucleoid associated stpA 2.6 KP1_4260 Cell surface structures
Prepillin peptidase dependent protein 2.6 KP1_0311
Two component system connector ycgZ 2.6 KP1_1728
Pullulanase-specific type II secretion system outer membrane lipoprotein
36
Table2 and 3. Significantly upregulated and downregulated genes by YjcC
overexpression. The selected genes show more than 2 log2 fold change (in absolute value) in the abundance of transcript between K. pneumoniae CG43S3[pRK415] andTable 2.3. Significantly downregulated genes by yjcC
overexpression
Proposed function Gene name Folda
expression
ORFb ID
Anaerobic response protein
Anaerobic class I fumarate hydratase fumB -5.7 KP1_2562
Transporter
Alanine/serine/glycine transport protein -6.8 KP1_2505
ABC transport system ATP binding protein -4.5 KP1_3173
PTS transporter subunits II ABC -5.0 KP1_3804
Methylgalactoside transporter inner membrane component mglC -4.8 KP1_0277 Methylgalactoside transporter system
Substrate-binding component
mglB -4.2 KP1_3815
Putative ABC transport system component -4.6 KP1_3175
Molybdate ABC transporter system -4.4 KP1_3995
Maltose/maltodextrin transporter ATP binding protein malK -4.2 KP1_0276
Sugar ABC transport system permease component -4.1 KP1_1424
Putative ABC transporter mocB -3.7 KP1_1423
Putative rhizopine uptake ABC transporter proY -3.7 KP1_1422
Permease
Putative PTS permease -6.5 KP1_0760
Putative amino acid permease -4.3 KP1_1204
Amino acid biosynthesis
Arginine succinyltransferase -4.5 KP1_2499
Acetylornithine transaminase -5.0 KP1_2498
Putative glutamine synthetase -3.3 KP1_2006
Energy/intermediary metabolism
Succinate antiporter -6.4 KP1_2563
Succinylarginine dihydrolase -5.3 KP1_2502
Glucosamine-fructose-6-phosphate
Succinylglutamate desuccinylase astE -4.0 KP1_2256
Phosphomannosemutase manB -3.4 KP1_3702
Putative monooxygenase subunit -3.1 KP1_1996
Putative acid phosphotase -3.1 KP1_3725
Regulators
DNA binding transcriptional repressor lldR -4.5 KP1_5297
Cell surface structures
Maltoporin lamB -4.8 KP1_0277
37
CG43S3[pJR1]. a. Log2
fold change represents the log
2 ratio of mRNA transcript levels of CG43S3[pJR1] to CG43S3[pRK415]. b. Open reading frame (ORF) ID is as annotated from K. pneumoniae NTUH K2044.38
Fig. 2.1. The yjcC is paraquat inducible, and SoxRS and RpoS dependent.
(A)
(A) The putative promoters respectively containing 525 bp (PyjcC1), 385 bp (PyjcC2) and 415 bp (PyjcC0) of yjcC were isolated and cloned into the LacZ reporter plasmid placZ15.
(B)
(B) The recombinant plasmids placz15, pPyjcC1, pPyjcC2 and pPyjcC0 were then transformed to K. pneumoniae CG43Z01 and the
-galactosidase activities of the
transformants grown to log-phase in LB broth were determined. The results are shown as an average of triplicate samples. Error bars indicate standard deviations.39
(C)
(C) Total RNA of K. pneumoniae CG43S3 was isolated after the bacteria were grown in 2 mM H2O2 or 30 μM of paraquat. Specific primer pairs used to detect the expression of soxR, soxS, rpoS, and yjcC are listed in Table 5.2. Relative fold expression was compared with the non-induced condition and determined by the 2-ΔΔCt method [207]. Error bars indicate standard deviation of the mean. Data are representative of three independent experiments.
(D)
(D) The expression of yjcC was determined in ΔsoxRS and ΔrpoS mutant by qRT-PCR.
Data are representative of three independent experiments, *, P< 0.001.
40
Fig. 2.2. Analysis of the deletion effects of yjcC upon exposure to oxidative stress.
(A)
(A) Diagrammatic depiction of the YjcC complementation plasmids is shown in the upper panel. The plasmid pJR2 is identical to pJR1 except that the E residue was replaced with A by site directed mutagenesis. The plasmid pJR3 carries only the EAL domain region of YjcC. Overnight cultures were collected and refreshed grown in LB until OD600 reached 0.6-0.7. 500 M paraquat or 10 mM H2O2 was then added and the cultures continued for 35 min and finally the cultures plated onto LB plates for colony formation.
41
(B)
(B) The phosphodiesterase activity of purify recombinant MrkJ protein, YjcC-EAL domain and YjcC-AAL domain proteins. BSA was used as a negative control. The activity is demonstrated using bis (pNpp) as substrate by the release of p-nitrophenol.
(C) (D)
(C), (D) Quantification of the c-di-GMP levels in K. pneumoniae CG43S3 using the ELISA kit according to the manual (Wuhan EIAab Science Co., Ltd). Three independent experiments were performed for the measurement Error bars shown are standard deviations, and asterisks indicate the differences with a statistical significance, P< 0.001.
42
Fig. 2.3. Deletion of yjcC places bacteria in an oxidative stress state.
(A)
(B)
(A) Cytoplasmic ROS content determination. (B) The oxidation determination of the cytoplasmic proteins and membrane lipids. The log-phased bacteria K. pneumoniae CG43S3[pRK415], ΔyjcC[pRK415] and ΔyjcC[pJR1] were exposed to 500
M
paraquat or 10 mM H2O2 for 40 min and the intracellular peroxide levels and the carbonyl groups were determined. Bars represent standard deviations (n= 4).43
(C)
(C) The DPPH radical scavenging activity measurement. The upper panel shows that, correlating with the antioxidant activity, the color of DPPH gradually changes from purple to yellow. Ascorbic acid and Butylated hydroxytoluene (BHT) were used as positive control. The lower panel shows quantitative measurement of the DPPH scavenging activity of the log-phased bacteria K. pneumoniae CG43S3[pRK415], ΔyjcC[pRK415] and ΔyjcC[pJR1]. Bars represent standard deviations (n= 4).
44
(D)
(E)
(D) SOD and (E) catalase activity determination as described in Materials and Methods. Left panels, in gel staining for the activity of Mn-SOD and Fe-SOD (D) and catalase HPI and HPIIs (E); Lanes 1, 2: CG43S3; 3, 4: ΔyjcC; 5, 6:
ΔyjcC[pRK415-pJR1]; 7, 8: ΔsoxRS; 9, 10: Δfur; 11, 12: ΔrpoS. Lanes 1, 3, 5, 7, 9, and 11 are protein extracts of the bacteria with no stress treatment; 2, 4, 6, 8, and 10 are protein extracts of the bacteria with paraquat. Right panels, quantitative measurement of the total SOD (D) and catalase activity (E).
45
Fig. 2.4. YjcC affects the CPS biosynthesis, biofilm formation and MrkA production.
(A)
(A) Sedimentation analysis (upper panel) and glucuronic acid content measurement (lower panel). Bacteria were grown overnight in LB broth at 37°C and subjected to centrifugation at 4000 rpm for 5 min. The glucuronic acid contents are expressed as the average of the triplicate samples. Error bars indicate standard deviations. *, P<
0.001 compared to the parental strain CG43S3 (n ≧ 3).Biofilm formation as assessed using crystal violet staining (upper panel) and spectrometry (lower panel).
46
(B)
(B) Bacteria were grown at 37°C in polystyrene plates for 24 h, the sessile bacteria stained with crystal violet, and then the stained cells eluted with 95% ethanol. *, p<
0.001. Data are representative of three independent experiments (triplicate in each experiment).
(C)
(B) Western blotting analysis for the expression of MrkA. Bacteria were grown overnight at 37°C with agitation in LB broth, and then total proteins extracted for western blot analysis. GAPDH was probed as a protein loading control.
47
Fig. 2.5. qRT-PCR analysis of the expression of mrkA, mrkH, and mrkI. Total
RNA of K. pneumoniae CG43S3[pRK415] and CG43S3[pJR1] were isolated after the bacteria were grown overnight in LB supplemented with 12.5µg/ml tetracycline.Specific primer pairs used to detect the expression of mrkA, mrkH, and mrkJ are listed in Table S2. Relative fold expression was compared with the non-induced condition and determined by the 2-ΔΔCt method. Error bars indicate standard deviation of the mean. Data are representative of three independent experiments.
48
Chapter 3
Transcriptome analyses of the cyclic di-GMP effect on the stress response in Klebsiella pneumoniae
CG43S3
49
3.1 Abstract
A comparative transcriptome analysis via RNA sequencing is employed to determine if the second messenger cyclic di-GMP plays a role in the pathogenesis of Klebsiella pneumoniae-associated liver abscess. The analysis reveals 94 upregulated
(>1.5-fold change) and 154 downregulated genes (< 2-fold change) with the increased production of diguanylate cyclase YdeH in K. pneumoniae CG43S3, a liver abscess isolate. The significantly activated genes included KP_0384 (perA), KP1_0385 (perC), ibpA, ibpB for heat shock response, pspB and pspD for phage shock response, and
mrkABCDF and mrkHIJ respectively for type 3 fimbriae expression and the fimbrial
regulation. Quantitative PCR analysis has been performed to validate the up-regulated genes which include ibpA, clpB, dnaK, grxA, dinI, mrkA, mrkH and mrkI. Compared to CG43S3[pRK415], the bacteria CG43S3[pRK415-ydeH] had increased the c-di-GMP levels and also the heat shock stress response, while decreased the acid stress and oxidative stress survivals.
3.2 Introduction
During the last decade, K. pneumoniae causing community-acquired primary pyogenic liver abscess (PLA) has become an emerging disease which receiving increasing attention. Many studies on the PLA pathogenesis have been reported [181-185], however, no conclusive points could be presented. The bacterial second
50
messenger c-di-GMP is a global signaling molecule that has been shown to influence a lot of physiological activity [186-188]. In recent year, its regulation on the stress response has been reported such as E.coli K12 YfgF, S. Typhimurium STM1344 in oxidative stress response[110,144], anti-toxin mqsA resistance of E. coli in acid stress[189], and V. cholera HapR in the stress response regulation [190]. However, c-di GMP regulation on the heat shock stress response has not been reported yet.
In the liver abscess isolate K. pneumoniae CG43, c-di-GMP has been shown as an effector for the expression of the PilZ domain protein MrkH and also the type 3 fimbriae expression [191]. Here we examine the effect of increasing c-di-GMP levels by transforming into K. pneumoniae CG43 with E. coli ydeH, which encodes a GGDEF domain protein and has been shown a high DGC activity [180,192]. A comparative transcriptome analysis of CG43S3[pRK415] and CG43S3[pRK415-ydeH]
using RNA-seq approach is then carried out in order to identify the genes responding to c-di- GMP regulation.
3.3 Results
3.3.1 The ydeH expression increased the c-di-GMP levels and biofilm formation
As shown in Fig.3.1A, the intracellular c-di-GMP content is increased 2.4-fold by the introducing the ydeH expression plasmid pRK415-ydeH into CG43S3. The biofilm forming activity analysis also supports the c-di-GMP mediated effect which is
51
to increase the biofilm formation activity (Fig. 3.1B). The results confirmed that the GGDEF domain protein YdeH carries a c-di-GMP DGC activity.
3.3.2 Up-regulated genes and down-regulated genes by the increase of c-di-GMP
levels
The comparative transcriptome analysis, on the basis of the genome annotation of K. pneumoniae NTHU-K2044 which is also a liver abscess isolate [112] revealed 19
genes significantly upregulated (> 2.3 fold expression) and 31 genes downregulated (< 4 fold expression). As shown in Table 3.1, the transcript levels of KP1_0384, KP1_0385, heat shock protein encoding genes ibpA, ibpB, clpB and psp as well as mrkH and mrkABCDF are increased. By contrast, the downregulated genes include
many metabolite transporters and permease (Table 3.2).
3.3.3 qRT-PCR analysis validates the c-di-GMP dependent expression manner
In order to confirm the RNAseq data, we have selected 8 genes including mrkA, mrkH, mrkI, grxA, ibpA, ibpB, clpB, and dnaK for qRT-PCR analysis. Fig.3.2 shows
mrkA and dnaK transcript levels are respectively increased 3.0 and 3.5 fold by the
increasing levels of c-di-GMP, other transcripts are increased at least 1 fold compared to wild type.
3.3.4 Increased c-di-GMP level upregulates the heat shock stress response but
downregulated the response to acid stress or oxidative stress
52
The transcriptome analysis implies a c-d-GMP dependent regulation of the stress response. The survival analysis against the stresses including heat (50℃), acid (pH 3), and oxidants (H2O2) revealed the increased c-di GMP level increased the heat shock survival rates but reduced the survival rates after bacteria exposure to 10 mM H2O2 or acid (Fig.3.3).
3.4 Discussion
In addition to mrkABCDF, mrkHI, and the stress response genes, the up-regulated genes also include KP1_0384 (perA) and KP1_0385 (perC) both coding for transcription factors. For enteropathogenic E coli, perA gene is a transcriptional activator carried by the virulence related perABC operon [193-196].
Gene annotation analysis of the CG43 genome reveled only perA and perC, implying per operon may play different role in regulation of the virulence in K.
pneumoniae. The dnaK transcript which is significantly increased by the increase of
c-di-GMP level encodes a molecular chaperone important for protein protection against stress [197,198]. The protein quality maintenance is essential for bacteria under exposure to stresses and therefore DnaK is extremely important for bacterial survival in harsh environment such as the infection site in the host body. Interestingly, phage shock related genes are also induced expression by c-di-GMP induced (Table 3.1). This implies the c-di-GMP level changes may be related to the bacterial resistance
53
to phage infection or to maintain the lysogenic state [199-201].
Table 3.1 and 3.2 Up-regulated genes and down-regulated genes from the comparison of CG43S3[pRK415-ydeH] and CG43S3[pRK415]
Proposed function Gene name Folda expression
heat shock chaperone IbpB
multiple drug resistance protein MarC KP1_2622 4.61
mrkA mrkA 4.48
sulfate/thiosulfate transporter subunit cysA 3.32
putative L-xylulose kinase lyx 3.25
putative oxidoreductase protein KP1_1109 3.20
molecular chaperone DnaK dnaK 3.14
ABC transporter ATP-binding protein KP1_5267 3.05
protein disaggregation chaperone clpB 3.02
recombination and repair protein recN 2.98
DNA-damage-inducible protein I dinI 2.97
LuxR transcription factor KP1_0384 2.94
putative acyltransferase KP1_5452 2.80
putative enzyme KP1_3482 2.74
sulfate adenylyltransferase subunit 2 cysD 2.69
thiosulfate transporter subunit cysP 2.62
putative methyltransferase KP1_1106 2.60
siroheme synthase cysG (NC_012731
4204327..4205743) 2.59 bifunctional putative transport protein/putative
kinase ydjN 2.50
phage shock protein B pspB 2.49
putative thioredoxin protein ybbN 2.47
Glutaredoxin I grxA 2.45
DNA binding protein, nucleoid associated htpG 2.43 carbamoyl phosphate synthase small subunit carA 2.41 PTS system, galactitol-specific IIC component KP1_1108 2.39 peripheral inner membrane phage-shock protein pspD 2.36
54
Table 3.2 downregulated
Proposed function Gene name Folda expression
putative glyoxalase/bleomycin resistance
protein/dioxygenas KP1_2502 -5.48
L-lactate dehydrogenase
lldD -5.47
putative PTS permease KP1_0762 -5.34
putative Glucosamine-fructose-6-phosphate
aminotransferase KP1_0765 -5.17
putative ABC transport system inner
membrane permease KP1_3174 -5.08
putative PTS permease KP1_0761 -5.04
putative NADH:flavin oxidoreductase KP1_2565 -4.96
succinylglutamate desuccinylase KP1_2504 -4.95
putative tartrate:succinate antiporter KP1_2563 -4.92 succinylglutamic semialdehyde
dehydrogenase astD -4.88
putative cytoplasmic protein yiiL -4.81
putative PTS permease KP1_0763 -4.77
D-alanine/D-serine/glycine transport protein KP1_2505 -4.71 putative glucosamine-fructose-6-phosphate
aminotransferase KP1_0764 -4.67
HP KP1_0341 -4.62
putative PTS permease KP1_0760 -4.62
putative ABC transporter KP1_1423 -4.61
putative rhizopine uptake ABC transport
putative phospho-beta-glucosidase KP1_3803 -4.45
putative ABC transport system periplasmic
binding component KP1_3175 -4.39
oxidoreductase KP1_1248 -4.32
sugar ABC transport system permease
component KP1_1424 -4.27
lysine/cadaverine antiporter cadB -4.27
anaerobic class I fumarate hydratase/fumarase
B fumB -4.25
putative myo-inositol catabolism protein iolB -4.23
maltoporin lamB -4.19
arginine succinyltransferase KP1_2499 -4.14
methyl-galactoside transport system
substrate-binding component mglB -4.13
citrate/acetate antiporter citW -4.12
putative ABC transport system ATP-binding
component KP1_3173 -4.10
bifunctional succinylornithine
transaminase/acetylornithine transaminase KP1_2498 -4.06
acetolactate synthase iolD -3.91
galactose-proton symport of transport system KP1_2730 -3.76
acetate permease actP -3.74
putative aldehyde dehydrogenase KP1_0552 -3.70
putative POT family di-/tripeptide transport
protein yjdL -3.66
putative aldehyde dehydrogenase KP1_1995 -3.66
55
putative amino acid permease proY (NC_012731
1688850..1690233) -3.43 glutamate/aspartate transport system permease gltJ (NC_012731
1578169..1578910) -3.39
putative inner membrane protein KP1_1483 -3.37
HP KP1_2985 -3.34
putative cytoplasmic protein KP1_0509 -3.30
Predicted sugar epimerase KP1_0551 -3.26
putative epimerase/isomerase KP1_0562 -3.25
putative di(mono)oxygenase alpha subunit KP1_1996 -3.23 type VI secretion system OmpA/MotB family
protein KP1_3385 -3.21
maltose ABC transporter periplasmic protein malE -3.17 glutamate and aspartate transporter subunit gltI -3.17
HP KP1_2314 -3.10
phosphomannomutase manB -3.09
pullulanase-specific type II secretion system
component J pulJ -3.09
HP KP1_0047 -3.09
aldehyde dehyrogenase astD (NC_012731
2168051..2169530) -3.06
putative glutamine synthetase KP1_2006 -3.04
putative PTS system transport protein KP1_1484 -3.01 PTS system beta-glucoside-specific
transporter subunits IIABC KP1_3804 -3.01
56
Fig.3.1. Cyclic di-GMP content. (A) C-di-GMP levels were determined using the
ELISA kit (Wuhan EIAab Science Co., Ltd). Three independent experiments were performed and error bars shown. (B) Biofilm formation. 20 µl of overnight bacteria were grown in LB at 37°C for 24 h. Biofilm development was observed visually as a function of pellicle formation and photographed. Data are representative of three independent experiments.57
Fig. 3.2 qRT-PCR analysis of the selected up-regulated genes. Total RNA were
isolated from the overnight culture of CG43S3[pRK415] and CG43S3[pRK415-ydeH].Specific primer pairs selected for the up-regulated genes with over 3-fold increase of expression. Relative fold expression was determined by the 2-ΔΔCt method. Data are representative of three independent experiments.
58