Molecular analysis of mutations in agrobacterium tumefaciens under selection pressure 2

a b Figure 3-17 a, the reaction that is catalyzed by SdhA inside A. tumefaciens. b, TCA cycle, the green cycle indicate the position in which SdhA function. 114 al. demonstrated that La protease, product of the lon gene, was an important determinant of transposase instability (1990). Later on, it was shown that the major nucleiod- associated protein H-NS protected structure related protein StpA from proteolysis by Lon protease since StpA was degraded relatively rapidly in E. coli H-NS mutant strain (Johansson and Uhlin, 1999). More recently, Rouquette et al. (2004) found that H-NS also promoted IS1 transposition by protecting its transposase InsAB’ from degradation by Lon protease. An apparently contradictory finding was that H-NS negatively regulated the stationary-specific sigma factor rpoS which was found to promote both point mutation and insertion mutation (Yamashino et al. 1995). The function of RpoS to transposition could be growth condition dependent while cellular rpoS content was much higher in hns mutant strain even under logarithmic growth phase in E. coli (Yamashino et al., 1995). In conclusion, our results suggest LonD could degrade the transposase from IS426 in A. tumefaciens, thus the transposition frequency of LonD mutant strain 715 was much higher than that of wild type. It could be interesting to decide the relationship of H-NS and LonD in A. tumefaciens. It seemed reasonable that the transposition was up regulated in all the three strains, A6340, 715 and Tcm3. However, it was still quite intriguing that how the cells “know” the point mutation does not work so that they must establish another set of mechanism to salvage themselves under certain condition. Did ChvG sense some signal in the environment so that it triggers further response? That was waiting to be confirmed. 115 It was likely that mutant 483 and Tcm5 mutate to tetracycline resistance in a different mechanism with A6340, 715 and Tcm3 since most of their mutations were point mutations instead of transposition on the minimal medium. Although the exact insertion mutation frequency of mutant 483 was higher (41.6×10-8) compared with that P P of wild type (6.7×10-8), the relative insertion mutation frequency (0.3% of total P P mutation) was very low because of the extremely high total mutation frequency. A gluconolactonase precursor gene involved in the glucose metabolism was disrupted in which showed dramatically higher mutation frequency compared with that of A6007. The enzyme gluconolactonase has been shown to be involved in the non-phosphorylated glucose degradation pathway (Figure 3-18). Compared with the common glycolysis, Embden-Myerhof Pathway, this pathway consumes more ATP. Interestingly, it happened that the deficient gene of Tcm5 Atu2583 encoding the enzyme glycosyltransferase was for the polysaccharide synthesis. These results indicated energy metabolism could be related to point mutation. Figure 3-6 clearly showed that the mutation hot spots were also different between A6007 and A6340 mutants. The hot spots of insertion in A6340 are 62 and 542 of tetR gene. On the contrary, the hot spot of insertion in A6007 tetR is 474. That further proved that A6007 and A6340 mutated to tetracycline resistance with different mechanism. Another system using mutations in the rpoB gene that yield the rifampicin 116 Figure 3-18: Non-phosphorylated glucose degradation pathway inside A. tumefaciens. The green cycle indicated the position in which the gluconolactonase function. 117 resistance (Rifr) phenotype was applied by us in order to analyze the point mutations P P of A. tumefaciens. As shown in table 3-6, again the mutation frequency to Rifr P P phenotype was much lower in A6340 compared with that of A6007. This further proved that ChvG may promote the point mutation. However, other strains which were susceptible to tetracycline on the rich medium did not show any difference with A6007 in the mutation to Rifr. It seemed that bacterial cells mutated to Tcr and Rifr with P P P P P P different mechanisms. The genes that affected the mutation to Tcr may not affect the P P mutation to Rifr. This was reasonable since Rifr was caused mainly by base substitution P P P P P P in the rpoB gene (Table 3-7; Wolff et al., 2004) instead of frameshift and insertion in the tetR system. We may think that sensor protein ChvG was at upstream of the mutational pathway since it could sense the environmental changes and transmit the signal to the cellular component. Sung and Yasbin (2002) also identified two-component system ComA and ComK involved in the regulation of differentiation in postexponential growth of Baccilus subtilis. The two-component system appeared to regulate aspects of stationary-phase mutagenesis in this bacterium. It was important to decide which genes were regulated by chvG-chvI two component system and if these genes were involved in mutational process. 118 Chapter 4. The role of ChvG in Agrobacterium tumefaciens 4.1. Introduction Our results clearly showed that ChvG, as the sensor protein of the two-component system, promoted the mutation to tetracycline and rifampicin resistance. It was reported that ChvG might function like a pH sensor and affected the expression of several acid-inducible genes like aopB and katA as well as some virulence genes like virB and virE in the Ti-plasmid (Li et al., 2002). However, no one has ever reported that ChvG exerted any effects to the mutational pathway. Moreover, chvG mutant strain is avirulent but we can not conclude if those acid inducible genes are the only reason that causes the avirulence of A. tumefaciens (Charles and Nester, 1993). In order to characterize the role played by ChvG during the mutational process as well as tumorigenesis, we performed experiments such as two dimensional PAGE gel electrophoresis and semi-quantitative PCR etc to identify the genes that are regulated by ChvG-ChvI two component system. 4.2. Materials and methods 4.2.1. Membrane proteins preparation The procedure of membrane proteins preparation followed the methods described by Maagd and Lugtenberg (1986) with proper modifications. Induced A. tumefaciens cells were collected and washed once with the induction medium (Pan et al., 1993) and twice with 50mM Tris-HCL, pH8.0, by centrifugation at room temperature. The combined cell pellet was resuspended in 50 mM Tris-HCL, pH8.5, 20% sucrose, 0.2 mM DTT, 0.2 mg/ml DNase I and 0.2/ml RNase A. The cell suspension was passed through a French press minicell (1000psi) three times. The cell lysate was treated with 0.2 mg/ml lysozyme 119 30 min, dilute with 10 ml 50 mM Tris-HCl, pH 8.0, 20% sucrose, mM EDTA . The cell suspension was incubated on ice for 30 and centrifuged at 3000 g for 15 at oC. P P All the remaining steps were carried out at oC. The resulting supernatant was further P P centrifuged at 12,000rpm for 15 and the supernatant was saved as the periplasmic fraction. The cell lysate was diluted with volumes of 50 mM Tris-HCl, pH 8.5 and centrifuged at 1,000 g for 20 min. The supernatant was then centrifuged at 150,000 g for hr. The supernatant was saved as the cytosolic fraction. The pellet was resuspended in 50 mM Tris-HCl, pH 8.5, 20% sucrose, 0.2 mM DTT and 0.2 M KCl by sonication. The resulting suspension was centrifuged at 150,000 g for hr. The supernatant was saved as the membrane wash fraction. The pellet (membrane proteins) was resuspended in 2.5 ml of mM EDTA, pH 8.0, 0.2 mM DTT and 20% sucrose by sonication. The suspension was layered onto a gradient containing a top layer of ml of mM EDTA, pH 8.0 and 53% sucrose over a bottom layer of ml of mM EDTA, pH 8.0 and 70% sucrose. The gradient was centrifuged at 150,000 g for 12 hr. The upper and lower bands were apparent and well separated from each other, and they were collected as the inner and outer membrane fractions, respectively. 4.2.2. Two-dimensional PAGE gel electrophoresis 4.2.2.1. Two-dimensional gel sample preparation The bacterial cells were cultured in IB 16-18 hours and then the cells were collected. The cells were suspend in 5ml 1×PBS. All the cells were subjected to times passages of Frech Press (1000 psi). The 60% TCA was added to the samples and wait on ice for one hour (1:4 v/v). The mixure was centrifuged at 20,000 ×g, 5min, 4oC. The P P supernatant was discarded and 1ml cold acetone was added to the pellet (keep in below -20 oC). The supernatant was poured out first and followed the second wash. We P P 120 used pipette for the 3rd wash. The pellet became loose after the third wash.We tried to P P use the pipette and not to pour away the pellet. The pellet was washed with acetone for three times. The supernatant was discarded; 150-200 µl cold acetone was added into each tube during each wash. Air dry the pellet at RT. The sample was dissolved with proper amount of lysis buffer (Bio-Rad Ready Prep Reagent plus TBP 2mM) for 30 at RT. The dissolve step was repeated for up to four times if the pellet still could not be solved. The sample was centrifuged at 14,000 rpm for 10 RT. Then the supernatant was collected as a sample. We measured the protein amount before keep in -80 oC. The preparation of membrane sample was shown in section 4.2.1. In the last P P step, the membrane proteins were dissolved in Reagent3. 4.2.2.2. Iso-electric focusing (IEF) The first-dimension isoelectric focusing was performed with the Ettan™ IPGphor™ Isoelectric Focusing System (Amersham, USA) according to the manufacturer's instructions. Briefly, the 18 cm Immobiline Drystrips with a linear gradient from pH range of to 10 or to (Amersham) were passively rehydrated for 12 to 16 h at room temperature with a mixture (340 µl) containing M urea, 2% (w/v) CHAPS, P P P P 0.5% immobilized pH gradient (IPG) buffer, 50 mM dithiothreitol, and a trace amount P P of bromophenol blue. After rehydration, 15 µl of rehydration buffer was added to the desired amount of protein sample (Total volume < 30 µl) and cup loading was employed to load the protein sample just prior to IEF. IEF was performed using the following conditions: 500 V for h, 4000 V for 1.5 h, 8000 V for 40000 Vh. 4.2.2.3. Second-dimensional PAGE Before running the second dimension, the 18 cm IPG strips (Amersham) were equilibrated in equilibration buffer I [6 M urea, 2% (w/v) SDS, 50 mM Tris-HCl pH 121 8.8, 30% (v/v) glycerol and 130 mM DTT, trace amounts of bromophenol blue] for 10 min, followed by equilibration buffer II [6 M urea, 2% (w/v) SDS, 50 mM Tris-HCl (pH 8.8), 30% (v/v) glycerol and 135 mM iodoacetamide (Sigma), trace amounts of bromophenol blue] for another 10 min. Equilibration buffer were stored in -80 oC. P P For second dimension SDS-PAGE, 12% polyacrylamide gels (20 x 20 cm, 1.0 mm thick) were used. The equilibrated-gel strips were placed on top of the 12% polyacrylamide gel and overlaid with 0.5% agarose (Seakem). The gels were mounted in a PROTEAN II XL Cell (Bio-Rad) and the Tris-glycine electrophoresis buffer [25 mM Tris, 250 mM glycine (pH 8.3), 0.1% (w/v) SDS] was added to the top and bottom of the gels between the glass plates. The proteins were then resolved with a constant current of 25 mA per gel for hours or so. 4.2.2.4. Silver Stain The gel was put into 50% methanol and 10% acetic acid; gently shake for 3-16h. Then wash it with 50% methanol alone for 15 min. Wash the gel with milli-Q water for and repeat for times. Then incubate the gel with sodium thiosulfate (fresh, 0.2g/L) for one minute. This step must be fast. Wash the gel with milli-Q water again one minute twice. Incubate the gel with silver nitrate (fresh, 0.2g/100ml in milli-Q; 200ml for one gel) for 25 min. Wash the gel with milli-Q water for one minute twice. Incubate the gel with sodium carbonate (30g/L) plus formaldehyde (250µl/L) for min, repeat 2-3 times depending on the developing time. Add EDTA sodium salt (14g/L) to stop the whole reaction and shake for 10min. Wash the gel with milli-Q water twice. 4.2.3. In-gel digestion The protein samples should be digested before Maldi-tof and MS. The in-gel 122 digestion process just followed the protocol from protein and proteomics centre, NUS. It is simply described as following. All the protein samples were adjusted to the same protein concentration and separated by 10% or 12% SDS-PAGE gel. Protein bands were carefully excised from silver-stained gels, cut into small pieces, and then destained with several washes of 50 mM ammonium bicarbonate in 50% aqueous acetonitrile. The gel pieces were dehydrated with 100% acetonitrile and dried in Savant Speed-vac. The dried gel pieces were rehydrated with a solution containing 10 mM DTT and 100 mM ammonium bicarbonate and incubated at 57°C for 60 to reduce the disulfide bonds. The reduced sulfhydryl group was alkylated by 55 mM iodoacetamide in 100 mM ammonium bicarbonate at room temperature for 60 min. After alkylation, the gel pieces were washed with 100 mM ammonium bicarbonate solution, dehydrated with 100% acetonitrile at least times, and then dried in Savant Speed-vac again. Sequencing grade modified trypsin (12.5 mg l-1 in 50 mM ammonium bicarbonate, pH 8.0) was added to the dried gel pieces and incubated overnight at 37°C. The resulting trypsinized peptides were extracted with 20 mM ammonium bicarbonate solution, 5% formic acid in 50% aqueous acetonitrile, and 100% acetonitrile, respectively. The extracted peptides were combined, lyophilized, and then resuspended in 0.1% trifluoroacetic acid (TFA). An aliquot of the resuspended peptides was spotted onto the stainless steel MALDI sample plate and overlaid with equal volume of matrix solution (20 g l-1 α-cyano-4-hydroxycinnamic acid in 0.1% TFA; 50% aqueous acetonitrile). The sample/matrix mixture was allowed to air dry. An Applied Biosystems Voyager-DE STR MALDI mass spectrometer was used to acquire the MALDI-time of flight (TOF) spectra. 123 References: Albright, L. M., Yanofsky, M. F., Leroux, B., Ma, D. and Nester, E. W. 1987. 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Plant J. 23: 1-28. 166 [...]... 1918.0, 524 4] 524 4.3 90 80 70 50 Mass (m/z) 29 69.4 35 12. 0 4700 Reflector Spec #1 MC[BP = 1918.1, 20 30] 20 30 .2 90 80 60 50 40 Mass (m/z) 29 69.4 35 12. 0 Figure 4-6: a, The spectra of A1(upper), A2(lower) 1 32 20 10 0 799.0 1341.4 1883.8 30 24 26 .2 2 426 .8 29 96.7705 26 69.5669 26 00.5613 25 58. 520 5 24 83.4504 24 99.44 82 2453 .29 42 1884 .2 2345.3777 22 61.1997 21 53 .28 86 40 21 35 .26 27 21 71 .21 80 22 11.3044 20 24.9431 100... 24 37.6543 1884 .2 2361.3784 22 61 .20 83 22 11. 322 0 1994.1830 1949.0433 1874.0555 1576.9576 1493.8767 1386.7980 13 42. 8375 30 21 72. 2158 22 11.3154 10 20 24.9543 1179.7 026 125 8.6898 1917.0430 100 1949.0 421 100 1917.0 427 1341.6 1873.0413 1674.1005 1 622 . 025 4 1576.9736 1474.9430 1386.7964 13 42. 8489 70 125 8.6873 0 799.0 123 4.7898 127 0.6943 % Intensity 60 127 0.6913 904.4860 10 904.4 924 8 42. 5 829 20 8 42. 5860 % Intensity... scores of A1, A2, A3, A4 and A5 are 1 62, 195, 1 62, 22 2 and 177 respectively 130 3 10 3 10 Figure 4-5: Two-dimensional PAGE gel electrophoresis of A6007 and A6340 membrane proteins Linear strip, pH 3-10 was used for IEF Left: A6340; right: A6007 131 20 0 799.0 1341.6 1884 .2 2 426 .8 24 26.8 30 12. 7805 30 24 83.4480 29 96.7683 26 28.5571 25 58.5347 24 99.4619 24 53 .29 22 2345.3843 21 53 .29 44 24 83.4553 40 25 58. 521 2 24 37.6543... 1341.6 1949.0317 30 12. 7839 26 69.5806 25 74.5330 24 99.4539 24 37.6 125 23 61.37 72 221 1.3135 21 44.1794 20 22. 9606 1946.0315 1949.0367 1664.9838 1576.9498 14 92. 8909 13 42. 8103 1386.7871 1414.7740 127 0.6876 1917.0380 90 1873.0370 1576.9 525 70 1480.9587 0 799.0 1386.7806 10 1343.8059 80 125 8.6638 8 42. 5640 % Intensity 125 8.6704 100 129 7.7731 904.4 725 20 830.5071 30 904.4796 40 831.4868 8 42. 5616 % Intensity 4700 Reflector... 799.0 1884 .2 2 426 .8 27 05.4731 25 80.5759 24 24.4480 22 72. 326 4 22 11.3306 20 00 .21 19 1874.04 72 1816.9943 1695.9041 1745.97 52 1619.9877 1544.0011 1480.8558 1347.7147 1341.6 1409.8480 1189.7084 10 1033.5911 20 128 1.7439 30 8 42. 5 829 % Intensity 60 29 69.4 35 12. 0 Mass (m/z) Figure 4-7: The spectra of protein A4 which is the hypothetical protein Atu4 026 134 Although the positions of A1, A2, A3 and A5 were different... comparing the membrane protein expression profile of wild type strain and A6340 even on the one dimensional gel Three bands were chosen for identification by MALDI-TOF Protein 1 and 3 were missed in the wild type sample while protein 2 was missed in the A6340 expression profile (Figure 4-8ab) As expected, number 2 protein matched to the hypothetical protein Atu4 026 , accession number gi|17937 722 , providing... by a pin brush (28 pins, 1 cm in diameter) or just scratched by a single pin Then appropriate volume of bacterial cell suspension (grown on MG/L plates for 2 days and suspended in MG/L at 2. 5 × 107 cells/ml) was inoculated onto each area of the wounds The plants were P P incubated in a growth room at 26 °C 124 4.3 Results 4.3.1 Results of two-dimensional PAGE gel electrophoresis As described in section... relationship of point mutation and insertion mutation The most significant finding of the project is the coupling of point mutation and insertion mutation of A tumefaciens mutants under tetracycline selective pressure A6340 (chvG mutant), Tcm3 (sdhA mutant) and 715 (lonD mutant) were demonstrated to have a very low frequency of mutation on MG/L rich medium compared with that of wild type strain A6007 However,... ChvG 140 5×104 cells WT ΔAtu4 026 1 A6340 ΔAtu4 026 8 Figure 4-11: Infection of A .tumefaciens cells on the leaves of Kalanchoe plants 5 × 104 in 2 l cell suspension of each strain (A6007, A6340 and ∆Atu4 026 ) were inoculated on the leaves for 17 days to test the ability of tumorigenesis of these strains Small tumors could be detected for A6007 Atu4 026 mutants but not A6340 inoculated leaves P P 141 4.3.5... protein Atu4 026 in the linear chromosome It was composed of 176 amino acids The molecular weight is 18.99 kD The molecular weight of the proteins on the two dimensional gel was almost accurate considering the position of AopB which exact molecular weight was 22 .79 kD To confirm this two dimensional gel result, we performed the one dimensional SDS PAGE gel electrophoresis The membrane proteins of A6007 . 20 30] 1917.0 427 125 8.6873 24 83.4480 8 42. 5860 1386.7964 1949.0 421 23 61.3784 1674.1005 20 24.9543 13 42. 8489 1474.9430 24 37.6543 127 0.6913 22 11.3154 25 58. 521 2 1873.0413 904.4860 1576.9736 1 622 . 025 4 21 72. 2158 30 12. 7805 799.0 1341.6 1884 .2 2 426 .8 29 69.4 35 12. 0 Mass ( m/ z ) 524 4.3 0 10 20 30 40 50 60 70 80 90 100 %. 29 54] 1917.0316 125 8.6638 21 53 .28 86 24 83.4504 8 42. 5616 22 61.1997 24 99.44 82 1949.0317 23 45.3777 1386.7806 25 58. 520 5 1343.8059 20 24.9431 831.4868 29 96.7705 24 53 .29 42 904.4 725 1480.9587 1576.9 525 22 11.3044 129 7.7731 1873.0370 21 71 .21 80 26 69.5669 26 00.5613 21 35 .26 27 . 524 4] 1917.0430 125 8.6898 24 83.4553 21 53 .29 44 23 45.3843 8 42. 5 829 1386.7980 1949.0433 29 96.7683 24 99.4619 25 58.5347 13 42. 8375 22 61 .20 83 1994.1830 904.4 924 22 11. 322 0 24 53 .29 22 127 0.6943 1493.8767 1874.0555 1179.7 026 1576.9576 123 4.7898 26 28.5571 133 Figure 4-6: b, The spectra of A3(upper),