Noirot-Gros et al. 10.1073/pnas.0506914103. |
Supporting Figure 7
Supporting Table 1
Supporting Figure 8
Supporting Figure 9
Supporting Table 2
Supporting Table 3
Supporting Table 4
Supporting Figure 10
Supporting Materials and Methods
Supporting Figure 7
Fig. 7. Phenotypic interacting assay of YabA mutants. (A) YabA mutant baits were confirmed for the specific loss of one interaction in a yeast two-hybrid assay. The different YabA mutant baits in the yeast haploid strain PJ69-4a (lines) were mated with PJ69-4a strains containing the preys YabA, DnaA, DnaN, TlpA, and AcuB (columns). Interaction phenotypes were monitored onto selective media as described in Materials and Methods. Mutants lacking the ability to grow onto SC-LUA are indicated by crosses of the corresponding colors. (B) A representation of the YabA interacting partners used in this study. Arrows represent interactions detected by the yeast two-hybrid assay and are oriented from bait to prey.
Supporting Figure 8
Fig. 8. Subcellular localization of GFP-YabA. A N-terminally fused to gfp–yabA construct was inserted at the ectopic amyE locus in a DyabA strain, so that it is the only copy of yabA in the cell. (A) Gallery of individual cells of increasing length containing one focus or two GFP-YabA foci (green). Membranes were stained with FM5-95 (red). When a single focus is present, it is localized at the cell center and divides as the cell extends, indicating that the YabA foci exhibit a dynamic localization in the cell. The foci are always associated with the nucleoid. A yfp–yabA fusion, inserted at the yabA chromosomal locus expressed from its native promoter, gave a similar localization pattern (5). (B) Colocalization of YFP–YabA and DnaX–CFP in living cells. Cells were grown to late exponential phase, mounted on agarose slides, and observed by using appropriate filter sets as described in Materials and Methods. YabA colocalized with DnaX, a component of the replisome, indiating that YabA localization at midcell corresponds to the position of the replication factory. (C) The distribution of the YabA foci in the cells was also compared with that of DnaX and that of Spo0J (used as an origin marker). We performed a statistical analysis of the intracellular position of the foci as a function of the cell length. The position of GFP-DnaX and GFP-YabA foci in cells containing one focus or two foci are represented in blue and pink, respectively. For cells containing GFP-Spo0J foci, the position of two and four foci are indicated in blue and pink, respectively. Comparison clearly shows that the YabA localization pattern is similar to that of DnaX, with one or two foci located at midcell and cell quarters, respectively, and clearly different from that of Spo0J, with two and four foci per cell. Together, these results allow us to conclude that YabA localizes in a replisome-like manner.
Supporting Figure 9
Fig. 9. Immunodetection of intracellular YabA derivatives. To compare the level of the YabA mutant derivatives relative to the wild type, we detected the intracellular YabA proteins in extracts by immunoblotting. The extracts were prepared from the same amount of cells, as described in ref. 6. The total cellular proteins of each strain were then fractionated by SDS/PAGE on 8% gels and transferred to a Hybond PVDF membrane (Amersham Pharmacia) by electroblotting using a semidry transfer system. Immunodetection was carried out by using an anti-YabA rabbit antiserum and a goat anti-rabbit IgG (or a G protein) horseradish peroxidase conjugate (from Bio-Rad). The image of the proteins was obtained with a Storm apparatus (Molecular Dynamics). The GFP- and YFP-YabA fusions have also been detected by using the anti-YabA antibody. Cell extracts were made from culture growing exponentially in LB medium supplemented with the appropriate antibiotics and xylose from the following B. subtilis strains (see Table 2): (A) Strains used in the origin visualization experiments (Fig. 4B) JJS39 (yabA+), JJS142 (DyabA), JJS138 (yabAN85D), JJS139 (yabAL86P), JJS137 (yabAV99A), and JJS49 (yabAL110P). Strain NIS6052 (yfp–yabA) was used to compare the relative levels of production of the yfp-fusion compare to the untagged yabA protein. (B) Strains used in the flow-cytometry experiments (Fig. 4A) NIS6050 (yabA::spec), NIS6052 (yfp–yabA), and derivatives yfp–yabAL86P, yfp–yabAN85D, and yfp–yabAL110P. (C) Strains used in the subcellular localization experiments (Fig. 3) JJS300 (gfp–yabA), JJS308 (gfp–yabAL86P), JJS306 (gfp–yabAN85D), JJS302 (gfp–yabAV99A), and JJS304(gfp–yabAL110P). (D) YabA levels in JJS331 cells carrying plasmid pDG148-yabA grown in LB supplemented with phleomycin (1 mg/ml) and various concentration of IPTG, as indicated.
Supporting Figure 10
Fig. 10. Complementation between YabA interacting mutants assayed by analysis of the relative cellular DNA content. The nucleoid DNA content analysis were performed as described in ref. 2. The DyabA or the yabA-Nim strains carrying a pDG148 plasmid or a derivative expressing either the wild-type YabA or YabA-Nim were grown exponentially in LB rich media supplemented with phleomycin (1 mg/ml) at 37°C. The cells were then fixed in EtOH 70% and stained with DAPI. The nucleoids were visualized by fluorescence microscopy on a Leica DMRA2 microscope. The images were acquired by using a Cool Snap HQ charge-coupled device camera (Roper Scientific/Princeton Instruments). The quantification of the DNA content in individual nucleoids was performed by using the metamorph software, and the data were processed with Microsoft excel. The relative DNA contents were estimated by using strain L5481 (dnaB19Ts flaD2) as internal standard in the digital image analysis, as described in refs. 4 and 7. The data were represented as the frequency of nucleoids (y axis) containing the relative numbers of chromosome equivalents (x axis), as determined from the count of 150–200 nucleoids for each strain. The results clearly show that the absence of yabA or the presence of only the YabA-Nim or YabA-Aim protein in the cell resulted in an increase of the relative DNA content in most nucleoids (≈70% of the nucleoids contain >2.5 chromosome equivalents (A, D, F). Note that nucleoids containing >10 chromosome equivalents were detected in these mutant strains. Upon expression of the wild-type YabA, >70% of the nucleoids contained <2.5 chromosome equivalents (B, E), with few nucleoids containing >8. The coexpression of both YabA-Nim and YabA-Aim mutants gave a nucleoid DNA content profile similar to that obtained with the wild-type YabA, indicating that these two interaction mutants can functionally complement (F). Note: The relative DNA contents given here represent an underestimation of the real DNA content as illustrated (7). Accurate DNA content in chromosome equivalents can be obtained by multiplying the experimental values by a factor of 1.3.
Table 1. Interacting mutants characterization
Mutation | Interaction phenotype | ||||
YabA | DnaA | DnaN | TlpA | AcuB | |
wt | + | + | + | + | + |
L13A* | + | + | + | – | – |
L24P | + | + | +/– | – | – |
L27A* | –† | – | – | – | – |
L41A* | + | + | + | + | + |
L52P | + | + | +/– | – | + |
Y83C | + | – | + | + | + |
N85D | + | – | + | + | + |
L86P | + | – | + | + | + |
V99A | + | + | – | + | + |
H100G* | + | + | – | + | + |
L110P | + | + | – | + | + |
C97R | + | – | – | + | + |
C97G* | + | – | – | + | + |
C109R | + | – | – | + | + |
C109G* | + | – | – | + | + |
C112R | + | – | – | + | + |
C112G* | + | – | – | + | + |
*From site-directed mutagenesis.
†
A residual growth can be observed after 10 da s. The expression of interaction phenotypes were monitored by growth of yeast colonies on selective media, as described in Materials and Methods. Phenotypes are +, Ade+ His+; +/–, Ade– His+; –, Ade– His–.
Table 2. Relevant genotypes of strains
Strain | Genotype | Source/construction |
168 | trpC2 | 8 |
JJS39 | 168 Dupp | 3 |
JJS142 | JJS39 DyabA | This work |
JJS137 | JJS39 yabA-V99A (yabA-Nim) | This work |
JJS49 | JJS39 yabA-L110P (yabA-Nim) | This work |
JJS138 | JJS39 yabA-N85D (yabA-Aim) | This work |
JJS139 | JJS39 yabA-L86P (yabA-Aim) | This work |
JJS300 | JJS142 amyE::Pxyl-gfp-yabA (SpcR) | This work |
JJS302 | JJS142 amyE::Pxyl-gfp-yabA-V99A (SpcR) | This work |
JJS304 | JJS142 amyE::Pxyl-gfp yabA-L110P (SpcR) | This work |
JJS306 | JJS142 amyE::Pxyl-gfp yabA-N85D (SpcR) | This work |
JJS308 | JJS142 amyE::Pxyl-gfp yabA-L86P (SpcR) | This work |
JJS338 | JJS142 amyE::Pxyl-cfp-yabA (SpcR) | This work |
JJS339 | JJS142 amyE::Pxyl-cfp-yabA-V99A (SpcR) | This work |
JJS340 | JJS142 amyE::Pxyl-cfp yabA-N85D (SpcR) | This work |
JJS341 | JJS339 yfp-yabA (CmR) | This work |
JJS342 | JJS339 yfp-yabA-N85D (CmR) | This work |
JJS343 | JJS340 yfp-yabA (CmR) | This work |
2641 | spo0J-gfp (KmR) | 9 |
JJS312 | 168 spo0J-gfp (KmR) | This work |
JJS310 | JJS142 spo0J-gfp (KmR) | This work |
JJS314 | JJS137 spo0J-gfp (KmR) | This work |
JJS316 | JJS138 spo0J-gfp (KmR) | This work |
JJS330 | JJS310 pDG148 | This work |
JJS331 | JJS310 pDG148-yabA | This work |
JJS332 | JJS310 pDG148-yabA-Nim | This work |
JJS333 | JJS310 pDG148-yabA-Aim | This work |
JJS334 | JJS314 pDG148 | This work |
JJS335 | JJS314 pDG148-yabA | This work |
JJS336 | JJS314 pDG148-yabA-Nim | This work |
JJS337 | JJS314 pDG148-yabA-Aim | This work |
JJS345 | JJS142 dnaX-cfp* (SpcR) | This work |
JJS347 | JJS346 yfp-yabA (CmR) | This work |
NIS6050 | CRK6000 DyabA::spec (SpcR) | 5 |
NIS6052 | CRK6000 DyabA::spec yfp-yabA (SpcR, CmR) | 5 |
MYA001 | CRK6000 DyabA::spec yfp-yabA-L110P (SpcR, CmR) | This work |
MYA002 | CRK6000 DyabA::spec yfp-yabA-N85D (SpcR, CmR) | This work |
MYA003 | CRK6000 DyabA::spec yfp-yabA-L86P (SpcR, CmR) | This work |
Standard techniques were used for the constructions (10, 11). The transformed strains were selected by plating onto LB nutrient agar containing the appropriate antibiotics: for E. coli, ampicillin, 100 µg/ml; for B. subtilis, spectinomycin, 60 µg/ml; kanamycin, 5 mg/ml; chloramphenicol, 5 µg/ml; and phleomycin, 1 µg/ml.
*The dnaX-cfp construct derived from strain SUXXX was kindly provided by P. Lewis (University of Newcastle, Callaghan, Australia).
Table 3. Plasmid constructs and oligonucleotides
Plasmids | Relevant genotype | Source/construction |
pDG148 | Pspac (ApR KmR PhleoR) | 12 |
pDG148-yabA | Pspac-yabA (ApR KnR PhleoR) | This work |
pDG148-yabA-Nim | Pspac-yabAV99A (ApR KnR PhleoR) | This work |
pDG148-yabA-Aim | Pspac-yabAN85D (ApR KnR PhleoR) | This work |
pSG1729-yabA | Pxyl-gfp-yabA (ApR SpcR) | This work |
pSG1729-yabA-Nim | Pxyl-gfp-yabAV99A (ApR SpcR) | This work |
pSG1729-yabA-Nim | Pxyl-gfp-yabAL110P (ApR SpcR) | This work |
pSG1729-yabA-Aim | Pxyl-gfp-yabAN85D (ApR SpcR) | This work |
pSG1729-yabA-Aim | Pxyl-gfp-yabAL86P (ApR SpcR) | This work |
pSG1190-yabA | Pxyl-cfp-yabA (ApR SpcR) | This work |
pSG1190yabA-Nim | Pxyl-cfp-yabAV99A (ApR SpcR) | This work |
pSMG05 | pCYB-dnaA (ApR) | This work |
pSMG28 | pTYB-yabA (ApR) | This work |
pTYB-dnaN | (ApR) | This work |
pCP112 | (ApR CmR) | 13 |
pCP112-yaat-yfp-yabA | yabA (ApR CmR) (PMH1) | 5 |
pCP112-yaat-yfp-yabA-Nim | yabA-L110P (ApR CmR) | This work |
pCP112-yaat-yfp-yabA-Aim | yabA-N85D (ApR CmR) | This work |
pCP112-yaat-yfp-yabA-Aim | yabA-L86P (ApR CmR) | This work |
Oligonucleotides
Names | Sequence |
mBD1ext | 5'-GTCTCCGCTGACTAGGGCAC |
mBD2ext | 5'-AGCTTCTGAATAAGCCCTCG |
yabA-F | 5'-ACTGGGCCCTTGGATAAAAAAGAGTTATTTGATACAGTC |
yabA-R | 5'-CGCGTCGACTTTTTTATTTAAGAATGACAGACAGAA |
Plasmid vectors were constructed in E. coli strain DH10B
Table 4. YabA-Aim complements the initiation defect of a yabA-Nim strain
Plasmid | pDG148 | pDG148/YabAwt | pDG148/YabA-Aim | pDG148/YabA-Nim | |||||||||
Strain | Foci per cell | 2–4 | 5–8 | >>8 | 2–4 | 5–8 | >>8 | 2–4 | 5–8 | >>8 | 2–4 | 5–8 | >>8 |
JJS142 (DyabA) | 32 | 18 | 50 | 65 | 17 | 18 | 10 | 10 | 80 | 35 | 17 | 48 | |
JJS137 (yabA-Nim) | 43 | 19 | 38 | 64 | 19 | 17 | 60 | 24 | 16 | 34 | 21 | 46 | |
Analysis of the distribution Spo0J foci in the cell population observed in Fig. 6. For each strain, at least 250 cells were analyzed and classified in three categories according to their Spo0J-GFP profile. Percentage of cell harboring: normal distribution, 2–4 foci per cell; slight overinitiation, 5–8 foci per cell; aberrant overinitiation, >>8 foci. In yabA+ strains, >98% of the cells fall into the first class.
Supporting Materials and Methods
Purification of YabA, DnaA and DnaN.
YabA, DnaA, and DnaN proteins were obtained by using the IMPACT system (NEB, Beverly, MA), in which each protein was C-terminally fused in frame with the self-cleavable intein-CBD affinity tag. YabA and DnaN protein fusions were expressed in Escherichia coli strains BL21 DE3 codon+, whereas DnaA was expressed in a dnaA-deficient strain derivative. The presence of the proteins through the purification steps was assayed by SDS/PAGE and Coomassie blue staining.Purification of YabA
. Intracellular production of YabA was achieved by first growing cells carrying plasmid pSMG28 at 30°C to an A600 of 1 in LB supplemented with thymine (25 mg/ml) and ampicillin (100 mg/ml). Then, 0.5 mM isopropyl b-D-thiogalactoside (IPTG) was added to induce the expression of the fusion protein, and the culture was further incubated overnight at 20°C. All subsequent steps were carried out at 4°C. The cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris·HCl, pH8, and 0.5 M NaCl), lysed by sonication, and centrifuged at 20,000 ×g for 1 h. The supernatant was loaded onto a 3-ml chitin column and washed with the same buffer. The affinity tag was then cleaved in the presence of 50 mM DTT according to the recommendation of the manufacturer. The cleaved untagged YabA was then eluted in buffer B (50 mM Tris·HCl, pH8, and 50 mM NaCl). The YabA-containing fractions were pooled and loaded onto a 1-ml HiTrapQ Sepharose column (Amersham Pharmacia Bioscience) previously equilibrated in buffer C (50 mM Tris·HCl, pH8, 50 mM NaCl, and 1 mM DTT). The bound YabA protein was eluted by a linear NaCl salt gradient (50 mM to 2M). The fractions containing YabA were then injected on a Superdex 200 HR10/30 column (Amersham Pharmacia Bioscience) formerly equilibrated in buffer D (50 mM Tris·HCl, pH8, 150 mM NaCl, and 1 mM DTT), and the purified YabA aliquots were stored at –20°C in the presence of glycerol 50%.Purification of DnaA.
The expression and purification of DnaA from cells containing plasmid pSMG05 were carried out essentially as described for YabA, except for the following modifications: (i) The expression upon IPTG induction was performed at 25°C for 7 h and (ii) a HiTrapSP column (Amersham Pharmacia Bioscience) equilibrated in buffer E (20 mM Hepes, pH 7.4, 50 mM NaCl, and 1 mM DTT) with a linear gradient from 50 mM to 2 M NaCl was used instead of a Superdex 200 HR10/30.Purification of DnaN.
Intracellular production of DnaN was achieved by first growing cells carrying plasmid pTYB-dnaN at 25°C for 5 h. The purification of DnaN was carried out essentially as described for DnaA.Determination of YabA Quaternary Structure.
YabA quaternary structure was determined by combining gel-filtration and sucrose-gradient sedimentation experiments (1) in buffer (50 mM Tris, pH7.5 and100 mM NaCl). With a Stokes radius (Rs) of 50 Å and a sedimentation coefficient (S) of 2.6 Sverdberg, we calculated a molecular mass of 56.7 kDa, corresponding to four YabA monomers (14.1 kDa). Furthermore, these experiments indicated that the YabA tetramer has an elongated shape.Random and Site-Directed Mutagenesis of yabA.
Site-directed mutagenesis of yabA was performed by PCR amplification and joining using oligonucleotides carrying the desired mutations. Random mutagenesis was achieved by PCR amplification under mutagenic conditions, where the concentration of one of the dNTP has been reduced from 200 to 40 mM. Amplification of the complete yabA coding sequence (357 bp) was performed by using the pGBDU-yabA bait plasmid as a template (2), and primers mBD1ext and mBD2ext (Table 3). These primers flank the cloning sites, and the PCR products were restricted by EcoRI + SalI, and gel purified. About 200 ng of the mutated fragments were then ligated with pGBDU-C1 vector. Next, the ligation mixture was subjected to 10 cycles of high-fidelity PCR amplification using external primers mBD1ext and mBD2ext, and the final product, containing the mutated yabA sequences flanked by 150-bp sequences homologous to pGBDU-C1, was gel purified. This material (about 1 mg) was mixed with a similar amount of pGBDU-C1 linearized by EcoRI + SalI digestion and cotransformed in the PJ69-4a haploid yeast strain. Transformants were selected on SC-U plates, and contained pGBDU-yabA mutant baits formed by gap-repair recombination.Screening of YabA-Interacting Mutants.
About 1,000 independent transformants were organized in 96-well format on SC-U plates to form a library of yabA mutant baits, including 6 that were generated by site-directed mutagenesis, and the library was mated with PJ69-4a strains containing the preys pGAD-yabA, pGAD-dnaN, and pGAD-dnaASID (amino acids 71-331) as described in ref. 10. Interaction phenotypes were scored by replica plating the diploids onto selective plates SC-LUH and SC-LUA. The YabA mutant baits affected for self-interaction or for interaction with DnaN or DnaA were unable to grow onto the SC-LUH and SC-LUA media. Diploids containing these YabA mutant baits were transferred onto a separate 96-well plate, and their impaired interaction phenotypes were confirmed. The mutations disrupting interactions were identified by sequencing yabA from the mutant bait in the corresponding haploïd cells.Construction of the GFP Fusions.
The N-terminally tagged GFP and CFP-YabA proteins were conditionally expressed from the xylose-inducible promoter Pxyl, from the ectopic amyE locus in the Bacillus subtilis chromosome. Primers yabA-F and yabA-R, carrying ApaI and SalI restriction sites, respectively, were used to PCR amplify the wild-type and mutant yabA genes from the corresponding bait vectors. The PCR fragments and pSG1729 were digested with ApaI + SalI, and pGS1190 was restricted by ApaI + XhoI. E. coli DH10B cells were transformed by the ligation mixtures, and single transformants were verified by sequencing. The gfp-yabA and cfp-yabA constructs were then integrated into the amyE locus of the B. subtilis DyabA strain (JJS142) by transformation, using spectinomycin (60 mg/ml) as a selection. The presence of the integrated gfp fusions were verified by PCR amplification from the amyE flanking region and by the inability of the strain to produce amylase.Construction of yabA Point Mutation B. subtilis Strains.
Point mutations were transferred in the chromosomal yabA locus by using the one-step gene-replacement procedure described in ref. 3. This methodology uses of the upp gene as a marker counterselectable by the 5'Fluorouracil (5FU). PCR-generated fragments containing yabA mutations (corresponding to the single amino acid changes L86P, N85D, L110P, and V99A in YabA) associated with the upp-cassette, were used to transform the 168 Dupp strain, as described in ref. 3. Homologous integration of the fragments leads to the replacement of the chromosomal yabA gene by the yabA::upp mutated copy and conferred 5FU sensitivity to the resulting strain. The precise excision of the upp-cassette yielded 5FU-resistant cells carrying a mutant yabA gene. The yabA gene was entirely sequenced in such strains to verify that only the desired point mutation was present.Fluorescence Microscopy.
For induction of the GFP- and CFP-tagged YabA proteins, cells from overnight cultures grown at 37°C in LB supplemented with spectinomycin (60 mg/ml) were diluted to OD600 = 0.01 in the same medium supplemented with xylose 0.2 to 0.5% until the OD600 reached 0.2–0.4, and were observed by using a fluorescence microscope Leica DMRA2. Cells coexpressing CFP-YabA and YFP-YabA were grown in selective LB medium, and cells were then streaked onto plates containing a low amount of xylose (0–0.1%) to limit induction of CFP-YabA and avoid quenching of the YFP signal. Cells coexpressing the DnaX-CFP and YFP-YabA proteins were grown in selective LB medium and observed at late exponential phase (OD > 0.5) for a better detection of DnaX-CFP. For microscopic observations, the cells were mounted on agarose slides as described in ref. 4. Images were acquired by using a Leica DC350F digital charge-coupled device camera. Appropriate filter sets to visualize the GFP, CFP, and YFP fluorescence signals were from Leica/WWR. DNA was stained with DAPI, and membranes were stained with FM5-95 (Molecular Probes).1. Marsin, S., McGovern, S., Ehrlich, S. D., Bruand, C. & Polard, P. (2001) J. Biol. Chem. 276, 45818–45825.
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