1.6 F IGURES
2.3.4 In vitro phosphorylation assay
To detect the Ser protein kinase activity, PA3346
L408-A571
and PA3347 were incubated in buffer containing 100 mM Tris-HCl pH 7.5, 50 mM KCl, 1.5 mM MgCl
2, 1 μCi [γ-32P] ATP or 2 mM ATP at 37°C for 1 h. The protein ratio of PA3346
L408-A571
and PA3347 was 1:1; as for in vitro phosphorylation assay of another putative anti-sigma antagonist protein PA2797, GST and GST-PA3347 proteins were used as negative control and positive control, respectively. GST-PA2797 (2
g) was
incubated with PA3346L407-A571 (1 g) and 1 mM ATP at 37oC for 10, 30 and 60 min.The reactions were quenched by adding the same volume of SDS-PAGE loading buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% sodium dodecyl sulfate, 0.1%
bromophenol blue, 10% glycerol, and 10% β-mercaptoethanol) and heated at 95oC for 10 min. The phosphorylation pattern was detected by autoradiography or Pro-Q®
Diamond phosphoprotein gel stain (Molecular Probes, USA). Pro-Q® Diamond phosphoprotein gel stain is an in-gel detection method of protein phosphorylation.
This fluorencence dye could detecte the phosphate group attached on Ser, Thr or Tyr residues (49,50) and the signals could be visualized by using a Typhoon 9200 Imager
(GE Healthcare) or a UV Box.
2.3.5 Protein pull-down assays
Glutathione agarose beads were suspended with the equal volume of PBS buffer (pH 7.4). Equal quantity of PA3347-GST (27.4
g) and PA3346
L408-A571-His6 were incubated with 500 μl of 50% slurry of glutathione agarose beads and 2 mM MgCl2 in the presence or absence of 2 mM ADP. After end-over-end mixing at 4oC for 2 h, the protein complexes were washed by 1 ml PBS buffer, eluted with 20 mM glutathione
39
(50 mM Tris-HCl buffer, pH 8.0) and analyzed on a 15% SDS- polyacrylamide gel.
As for the protein-protein interaction assay of PA3346-GST and RpoN-His6, the experimental procedures were the same as described above.
2.3.6 Western blotting analysis
The PA3347-GST and PA3346L408-A571-His
6 proteins on the 15% SDS- polyacrylamide gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare) at 100 V for 1 h. The blot was blocked in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) containing 5%
skim milk at room temperature for 1 h. Then, the blot was incubated with a monoclonal anti-GST antibody (6G9B9, Novus Biologicals, USA) or a monoclonal anti-His antibody (ab18184, Abcam, UK) at 4oC overnight. After 10-min washes with PBS buffer for three times, a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) was applied to the blot and the incubation was done at room temperature for l h. Finally, the signals of the target proteins were revealed by HRP colorimetric assay (Vector® NovaREDTM Substrate kit, Vector Laboratory, USA).
2.3.7 In vivo protein-protein interaction assay by GFP fragment reassembly
The vectors used in this study were provided as a gift by Professor Lynne Regan of Yale University (29). Gene PA3347 used as the bait and PA3346 (1222-1713) used as the prey were cloned into pET11a-link-NGFP and pMRBAD-link-CGPF, respectively. The two plasmids were co-transformed into E. coli BL21 (DE3) and cultured in LB medium containing 100 μM ampicillin and 35 μM kanamycin at 37oC for 12 to 16 h. To perform the GFP fragment reassembly, ten microliters of the overnight cultures were applied to the LB agar containing the same antibiotics, 10040
μM IPTG and 0.05% arabinose. Then, the plates were statically incubated at 20°C for 2 to 3 days. The bacteria cells grown on the plates were re-suspended with PBS buffer and examined under an upright fluorescence microscope (BX-51; Olympus) connected with a CCD camera. The data were analyzed by using the SPOT Advanced Plus Imaging software (Sterling Heights, MI). In the case of searching the putative factors interacting with PA3346408-571, all possible genes were cloned into pET11a-link-NGFP and pMRBAD-link-CGPF, respectively. The experimental procedures were the same as described above and the detail information about the possible genes was listed in Table 2.6.3.
2.3.8 Chrome azurol S (CAS) plate assay
The CAS agar plates were prepared on the basis of method described by Schwyn and Neilands (51). Because the preparation for the plates is difficult, we also refer to the step-by-step protocol published by Louden and Lynee (52). The CAS plate contains 0.1 mM CAS, 0.2 mM HDTMA, 10 M FeCl3．6H2O, 100 mM PIPES, M9
minimal salt (Sigma-Aldrich®), 0.1 mM CaCl2, 2 mM MgCl2, 0.3% Casamino acid, 0.2% glucose and 1.5% agar. The pH was adjusted to 5.6 with NaOH. P. aeruginosa PAO1 wild type and mutant strains were grown overnight in LB medium at 37°C, and then 3 l of the culture was inoculated onto the CAS agar plate. After 24 h incubation at 37°C, the siderophore production of each bacterial strain was compared according to the orange halo around the colonies.
2.3.9 N-Acyl homoserine lactones (AHL) reporter plate bioassays
The overnight LB culture of C. violaceum CV026 was added into the semi-solid LB agar (0.3%, w/v) in the volume ratio of 1:100, mixed well and then poured immediately on the surface of NB, LB or M8 agar plate (1.5%, w/v). After the upper
41
layer agar solidified, the 200 l pipette tips put inversely on the 1.5% agar plate were removed to form wells on the overlaid agar. Each of the wells was filled with 10 l overnight LB cultures of P. aeruginosa PAO1 wild type or mutant strains, respectively.
Then, the plates were kept in the upright position and incubated at 30°C for 24 h. C.
violaceum CV026 is a mini-Tn5 transposon insertion mutant of C. violaceum ATCC
31532, causing a violacein-negative strain (53,54). This purple pigment (violacein) production can be restored by providing the exogenous AHLs. Therefore, the AHLs produced by P. aeruginosa strains could be measured qualitatively by examining the purple pigment formed around the wells.2.3.10 Congo red plate assay
Plates contain Tryptone (10 g/L), Congo red (40 mg/L) and 1% Bacto agar (55,56). Bacteria were inoculated on the surface of the plate by using a sterile toothpick. After 2 days of incubation at 30°C, the colony morphology of each strain was recorded with a Canon camera (PowerShot G10). The assays were performed
The protein structure of B. stearothermophilus SpoIIAB, a Ser protein kinase and an anti-sigma factor, has been solved. It is revealed that SpoIIAB is structurally distinct to the Hank’s type Ser protein kinases (57) while exhibiting high similarities to bacterial two-component histidine kinases and ATPases of the GHKL superfamily (58). SpoIIAB contains three of the four conserved regions in GHKL superfamily, motif I, II and III which corresponded to the N, G1 and G2 boxes in histidine kinase
42
(58). The C-terminal region of PA3346 contains a histidine-like or Hsp90-like ATPase domain, which belongs to the GHKL superfamily. Sequence alignment performed by VetcorNTI shows that PA3346L408-A571 also has N (EX3
N
X3H, x reveals any amino
possesses a Ser protein kinase activity toward PA3347
According to the preliminary amino acid sequence analysis, it is possible that PA3346
L408-A571
can phosphorylate PA3347. Therefore, the C-terminal region of PA3346 was cloned and then purified using Ni
2+
-charged affinity chromatography.
The purified PA3346
L408-A571
protein was incubated with an equal amount of PA3347 and 1 μCi [γ−32P] ATP at 37°C. Fig. 2.6.2 clearly demonstrates that the intensities of PA3347 phosphorylation signals increase over time, indicating that PA3346
L408-A571
possesses a protein kinase activity. Besides, the phosphorylation site on PA3347 indeed locates at Ser-56 because a PA3347 variant with a S56A mutation could not be phosphorylated (Fig. 2.6.3) (34). More interestingly, PA3346
L408-A571
is divalent cation dependence (Fig. 2.6.4). It can utilize at least three kinds of ions and the enzyme activities were in the order of Mg
2+
2.4.3 PA2797 is not the substrate of PA3346
L408-A57143
Except PA3347, the hypothetical protein PA2797 is predicted as an anti-sigma factor antagonist based on the result of InterProScan. For further investigation, we also analyzed the amino acid sequence identities and similarities of PA2797 (NP_251487) with PA3347 (NP_252037), B. subtilis SpoIIAA (NP_390228) and B. subtilis RsbV (NP_388352). Although PA2797 shared only about 10% sequence identity with PA3347, SpoIIAA and RsbV, a conserved LX6
DS
X2LG motif (Fig. 2.6.6), where X is
any amino acid and S is the target phosphorylation site of Ser protein kinase, was found. Furthermore, PA2797 and PA2798 are the homologs of B. subtilis SpoIIAA and SpoIIE. PA2797-PA2798 may form another partner switching system in P.aeruginosa PAO1 but lack a specific Ser protein kinase and cognate sigma factor. A
recent study also revealed that P. aeruginosa PA2797 and PA2798 mutants exhibited an enhanced susceptibility to aminoglycosides (59). Whether PA2797 regulates a yet to be identified sigma factor which confers the bacterial resistance against aminoglycoside through phosphorylation by PA3346408-571 remains to be clarified.Therefore, we have cloned and purified PA2797 and included the protein in an in vitro phosphorylation assay. As shown in Fig. 2.6.7, both ovablumin and PA3347 incubated with PA3346408-571 and ATP displayed strong fluorescence signals, whereas GST as a negative control did not. GST-PA2797 yielded weak fluorescence signal in the assay, although the signal intensities of GST-PA2797, GST-PA2797 incubated with PA3346408-571 in the absence or presence of ATP showed no obvious difference. The result indicates that PA2797 might not be phosphorylated by PA3346408-571. Thus, PA3347 is the only substrate of PA3346408-571 found. The orphan anti-sigma factor antagonist PA4452 (NP_253142) was not further investigated in this study.
2.4.4 PA3346
L408-A571
interacts with PA3347 in the presence of ADP in vitro
Previous literatures have reported that ADP-containing SpoIIAB and SpoIIAA44
formed a stable protein complex (36,60). PA3346L408-A571 and PA3347 may behave like an anti-sigma factor and an anti-sigma factor antagonist, respectively. Whether the two proteins can interact with each other was investigated using the GST-pull down assay. The purified PA3347-GST and PA3346
L408-A571
-His6 were tested for their binding in the assay and the effect of ADP in protein-protein interaction was also determined. Our finding indicates that in the presence of ADP, PA3346
L408-A571
could be co-eluted with of PA3347 (Fig. 2.6.8). Comparison of the phophorylation levels of the freshly purified, calf intestine alkaline phosphatase (CIP) treated, and the fully phosphorylated form of PA3347, it could be concluded that about 10% of the purified
PA3347 proteins produced in the heterologous host E. coli BL21 (DE3) were phosphorylated, indicating that most of PA3347 proteins are unphosphorylated
(Fig.2.6.5). These results reveal that PA3346
L408-A571
can bind to the unphosphorylated form of PA3347 in the presence of ADP.
2.4.5 PA3346
L408-A571interacts with PA3347 in vivo
To further verify whether PA3346 L408-A571 interacts with PA3347 in vivo, a GFP fragment reassembly assay was performed. The DNA fragment containing the coding region of PA3346L408-A571 was cloned into pMRBAD-link-CGFP to produce a recombinant protein fused to C-terminal fragment of GFP as the prey. The entire coding region of PA3347 was cloned into pET11a-link-NGFP to produce an NGFP- PA3347 fusion protein as the bait. The protein-protein interaction of the bait and prey can lead to the GFP fragment reassembly (17,29). E. coli BL21 (DE3) cells harboring pMRBAD-Z-CGFP and pET11a-Z-NGFP were used as a positive control. As shown in Fig. 2.6.9, the bacterial cells of the positive control and PA3346408-571-CGFP and
45
NGFP-PA3347 pair displayed green fluorescence signals under the fluorescence microscope at an excitation wavelength of 488 nm, whereas no fluorescence emission was detected in negative control cells. The result indicates that PA3346408-571 interacts with PA3347 in vivo.
2.4.6 PA3346
L408-A571cannot interact with RpoN in the presence of ATP in vitro
Our data indicated that PA3346L408-A571 possessed a Ser protein kinase activity and interacted with the anti-sigma factor antagonist PA3347 in vitro and in vivo, suggesting that PA3346L408-A571
may behave as not only a Ser protein kinase, but also an anti-sigma factor (36,45,47). Like SpoIIE-SpoIIAB-SpoIIAA-F in B. subtilis, PA3346-PA3347-unknown sigma factor in P. aeruginosa PAO1 might form a partner switcher regulatory system. To clarify if PA3346
L408-A571
is an anti-sigma factor, it is important to find the unknown sigma factor. In a previous study, it has been proved that the HptB-PA3346-PA3347 cascade regulated the bacterial motility and biofilm formation (34,35,56). In addition, RpoN (54) has been reported to be required for the flagella biogenesis and bacterial motility in P. aeruginosa (61), Xanthomonas
campestris (62), Vibrio fischeri (63) and E. coli MG1655 (64). The rpoN mutant of P.
fluorescens CHA0 displayed reduced swimming and swarming motilities (65). Based
on these studies and the results of our phenotype assays, we proposed that RpoN is the possible sigma factor interacting with PA3346L408-A571. Whether RpoN and PA3346L408-A571 interact with each other is investigated by using a GST-pull down assay. The purified RpoN-His6 and GST-PA3346were tested for their binding in the presence of 1 mM ATP (66). Fig. 2.6.10A shows that RpoN-His6 and a small amount of GST-PA3346L408-A571
were eluted with PBS buffer. When washed by 20 mM
46
glutathione buffer, only GST-PA3346L408-A571 proteins were eluted (Fig. 2.6.10B). The result indicates that RpoN-His6could not be co-eluted with GST-PA3346 L408-A571 in the presence of ATP. Therefore, RpoN might not be the sigma factor we are searching for.
2.4.7 ΔhptB mutant strain displays a higher production level of exopolysaccharides
In our previous study, analysis of PA3346 and PA3347 gene knock-out mutants revealed that HptB-mediated signaling pathway is associated with biofilm formation (34). Recently, some studies demonstrated that polysaccharides contribute to biofilm formation in nonmucoid strains (67,68). Therefore, we conducted the Congo red plate assay to determine whether the biofilm formation is linked to the production of exopolysaccharides. Congo-red dye has been shown to bind extracellular matrix components, especially the exopolysaccharides. When grown on the Congo red plate, the ΔhptB mutant (MPA45) colony was completely dark pink, indicating more polysaccharide production. In contrast, PAO1, MJL46 and MJL47 displayed the similar colony morphology. The periphery of PAO1, MJL46 and MJL47 colonies was dark pink and the central was pale white, revealing less polysaccharide production (Fig. 2.6.11). The data is consistent with that of P. aeruginosa PAK strain studied by a research group in CNRS, France (56). Thus, based on the result of Congo red plate assay, the biofilm formation regulated by HptB-mediated pathway is in relation to the exopolysaccharide production.
2.4.8 No significant phenotype differences are found in CAS plate assay and AHL reporter assay
There are 24 sigma factors found in P. aeruginosa PAO1 (69). In order to know
47
in advance which sigma factors might participate in the HptB-mediated partner switching regulatory module, we performed the AHL reporter assay and CAS plate assay first. RhlR/RhlI quorum sensing system can regulate the bacterial swarming motility by activating the gene transcription of rhlAB and rhlC when the concentration of C4-HSL reaches a threshold (70,71). Moreover, stationary-phase
factor RpoS
negatively regulates the transcription of rhlI whose gene product is responsible for the synthesis of C4-HSL (72). It is interesting to clarify if RpoS regulates the swarming motility through the C4-HSL-mediated pathway (42,73,74). Using the AHL reporter assay to determine whether P. aeruginosa PAO1 wild type and mutant strains produced different amounts of C4-HSL, we found that regardless the culture medium, (NB, LB or M8 agar) , the level of purple pigment produced by the reporter strain C.violaceum CV026 shows no significant difference (Fig. 2.6.13). The result indicates
that P. aeruginosa PAO1 and mutant strains produced comparable amounts of C4-HSL. This study also interested in PvdS, aECF factor controlling the
transcription of pyoverdine biosynthesis genes (75,76). If PvdS participates in the partner switching regulatory system, the Fe3+ chelating abilities would be different between P. aeruginosa PAO1 wild type, MPA45, MJL46 and MJL47 strains. However, the result of CAS plate assay reveals that the diameters of orange halos around the bacterial colonies are similar (Fig. 2.6.12), indicating that the iron chelating ability of the strains tested has no difference. In summary, RpoS and PvdS may not participate in this HptB-mediated partner switching regulatory system.2.4.9 Is PA3346
L408-A571an anti-sigma factor?
To further investigating the factors interacting with PA3346, a GFP fragment reassembly assay was performed. The genes encoding the sigma factors including RpoH, RpoD, RpoS, RpoN, FiuI, AlgU, SigX, FpvI, PvdS, the uncharacterized ECF
48
factors PA1351, PA2896, PA3285, the putative54-dependent transcriptional regulators FleQ, PA1663, PA2359 (69), putative 54 modulation protein PA4463 and a regulatory protein RsmA (77) are cloned into pMRBAD-link-CGFP or pET11a-link-NGFP, respectively. The plasmids used in this assay are co-transformed into E. coli BL21 (DE3) with the PA3346-link-NGFP or PA3346-link-CGFP. The bacterial cells containing the bait and prey vectors are cultured on LB agar plates with appropriate antibiotics and inducers for 3 days at 20oC and then examined under a fluorescence microscope. Although we have tested about 73 combinations of the bait and prey plasmids, the bacterial cells display no fluorescence signal other than the positive control. This result may be due to the conformational incompatibility between the bait and prey GFP fusions or no protein-protein interaction between these factors and PA3346.
Our preliminary result indicates that PA3347 and PA3346L408-A571 form a stable protein complex in the presence of ADP. Besides, the phosphorylation of PA3347 by PA3346 leads to the release of PA3347~P from the protein complex (78), agreeing with the mechanism of partner-switching regulatory module that the phosphorylation state of the anti- antagonist is an inactive form (47,66,79). The findings reveal that PA3346 might be a SpoIIAB-like anti-sigma factor antagonized with a sigma factor.
However, we failed to find the corresponding sigma factor in the GFP fragment reassembly assay. It is possible that PA3346 may function in a manner different from the traditional partner switching system described for B. subtilis and other Gram-positive bacteria. For instance, it is reported that the partner switching cascade BtrU-BtrV-BtrW regulates the type III secretion (TTS) in Bordetella bronchiseptica, a Gram-negative bacterium causing infectious bronchitis. The authors of this study found that the btr partner switching module does not influence the transcription of the
49
TTS genes but is essential for the regulation of TTS (37). Thus, they proposed that the switching proteins associated with free BtrW (predicted as an anti- factor) could be a part of the TTS machinery or another regulatory molecules, but not a sigma factor (47). Furthermore, a similar partner switching system was found in a Gram-negative pathogen Chlamydia trachomatis (RsbU-RsbV1-RsbV2-RsbW). No successful experiment results support that C. trachomatis RsbW could bind with
66, 54 or its most likely target 28. Therefore, the authors consider that the target of C. trahomatis switching protein is not a sigma factor but instead is another protein which controls the response functions post-translationally (80). These studies suggest that the switching proteins associated with SpoIIAB-like protein are different from the partner switching system in B. subtilis. PA3346 may be associated with a transcriptional factor or a regulatory factor which could be a part of flagella machinery or exopolysaccharide export system. Hence, HptB-PA3346-PA3347 regulatory cascade could regulate the swarming motility and biofilm formation either transcriptionally or post-translationally. There are 5571 open reading frames (ORFs) and at least 550 transcriptional regulators identified in P. aeruginosa PAO1 genome (69,81). For the high-throughput screening of the corresponding factors participated in the partner switching regulatory module, it is required to construct a P. aeruginosa genome fragment library by using the yeast two-hybrid system (82), bacterial two-hybrid system (83,84) or the bimolecular fluorescence complementation (BiFC) method (29).Based on the previous work and this study, we suggest that the bacterial swarming motility and biofilm formation are under the regulation of HptB-mediated signaling pathway. The output responses might be controlled through the protein-protein interaction of PA3346L408-A571 with either PA3347 or an unknown regulatory factor. Our hypothetical model (Fig. 2.6.14) shows that PA3347, an
50
anti-sigma factor antagonist, is inactivated by phosphorylation on Ser-56 through the action of Ser protein kinase (PA3346L408-A571). This would lead to the disassociation of PA3346L408-A571 from PA3347~P and allow it to bind to the corresponding regulatory factor; When the receiver domain of response regulator PA3346 is phosphorylated, the Ser protein phosphatase activities are increased and result in the dephosphorylation on PA3347 Ser-56. PA3347 is active and could bind to PA3346L408-A571 to release the corresponding regulatory factor. Studies are underway to investigate which protein factors participate in this regulatory system. If the model comes out to be true, it might be a novel combination system of two-component system and partner switching regulatory module in Gram-negative bacteria.
51
2.5 Tables
Table 2.5.1 Bacterial strains and plasmids used in this study
PAO1 Nonmucoid wild type strain Laboratory stock
MPA45 PAO1 ΔhptB Laboratory stock
MJL46 PAO1 ΔPA3346 Laboratory stock
MJL47 PAO1 ΔPA3347 Laboratory stock
C. violaceum CV026 a mini-Tn5 transposon insertion mutant of C. violaceum ATCC 31532, violacein-negative strain Laboratory stock Plasmids
pET30a Kmr, His tag protein expression vector Novagen
pGEX-5X-1 Apr, GST tag protein expression vector GE Healthcare
pET11a-Z-NGFP Apr, plasmid vector that expresses a fusion of an antiparallel leucine zipper peptide to NGFP (29) pET11a-link-NGFP Apr, plasmid vector designed for fusion of a target protein to the N-terminal fragment of GFP (1-157) (29) pMRBAD-link-CGFP Kmr, plasmid vector designed for fusion of a target protein to the C-terminal fragment of GFP (158-238) (29) pMRBAD-Z-CGFP Kmr, plasmid vector that expresses a fusion of an antiparallel leucine zipper peptide to CGFP (29)
pET11a-Z-NGFP Apr, plasmid vector that expresses a fusion of an antiparallel leucine zipper peptide to NGFP (29) pET11a-link-NGFP Apr, plasmid vector designed for fusion of a target protein to the N-terminal fragment of GFP (1-157) (29) pMRBAD-link-CGFP Kmr, plasmid vector designed for fusion of a target protein to the C-terminal fragment of GFP (158-238) (29) pMRBAD-Z-CGFP Kmr, plasmid vector that expresses a fusion of an antiparallel leucine zipper peptide to CGFP (29)