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An Expanded View of Bacterial DNA Replication

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An Expanded View of Bacterial DNA Replication An expanded view of bacterial DNA replication Marie-Franc¸oise Noirot-Gros*, Etienne Dervyn*, Ling Juan Wu†, Peggy Mervelet*, Jeffery Errington†, S. Dusko Ehrlich*, and Philippe Noirot*‡ *Ge´ne´ tique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France; and †Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom Edited by Richard M. Losick, Harvard University, Cambridge, MA, and approved April 22, 2002 (received for review January 23, 2002) A protein-interaction network centered on the replication machin- pGBDU bait vector (URA3), and the hybrid plasmids were ery of Bacillus subtilis was generated by genome-wide two-hybrid isolated from Escherichia coli and transformed into PJ69–4a screens and systematic specificity assays. The network consists of yeast strain (8). DNA sequences of all cloned fragments were 91 specific interactions linking 69 proteins. Over one fourth of the verified. The yeast strain PJ69–4␣ was a gift from P. James. For interactions take place between homologues of proteins known to trihybrid experiments, a p3H vector (TRP1) was generated by interact in other organisms, indicating the high biological signifi- deleting the GAL4 DNA binding domain (BD) from pGBD cance of the other interactions we report. These interactions vectors (8) and replacing it with the nuclear localization signal provide insights on the relations of DNA replication with recom- from simian virus 40 T antigen and an S-tag sequence (Nova- bination and repair, membrane-bound protein complexes, and gen). The media for growth of E. coli, B. subtilis, and Saccha- signaling pathways. They also lead to the biological role of un- romyces cerevisiae were as described (9–11). known proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interac- Library Construction. Three B. subtilis genomic libraries (BSLs), tion map provides a valuable tool for the discovery of aspects of BSL-C1, BSL-C2, and BSL-C3, were constructed in E. coli bacterial DNA replication. (DH10B, Life Technologies, Cergy Pontoise, France) strain as described previously (8). Restrictions of the 4.2-megabase B. he replication of the bacterial chromosome is carried out by subtilis chromosome produced Ϸ1.6 ϫ 105 DNA ends that could Ta large multiprotein machine, the replisome, in which the be ligated into the pGAD prey vectors. Each library contained activities of individual polypeptides are highly coordinated to at least 2.5 ϫ 106 clones, thus providing a 15-fold redundancy. achieve efficient and faithful DNA replication. The components The PJ69–4␣ yeast strain was transformed by each library DNA of bacterial replisomes have been characterized extensively, as described (12), and at least 1.5 ϫ 107 prey-containing colonies revealing the molecular mechanisms at work in a DNA- were harvested and pooled. Aliquots containing Ϸ1 ϫ 108 replication apparatus (1, 2). Localization studies indicated that transformed yeast cells were stored at Ϫ80°C. the replication machinery is preferentially at midcell, suggesting a factory model of replication in which the DNA template moves Library Screening. A mating strategy derived from that described through a rather stationary polymerase (3). In contrast, the in ref. 13 was used. For each screen, one BSL-C1, BSL-C2, and origin regions of the chromosomes are moving toward the cell BSL-C3 vial was thawed, and cells were mixed with PJ69–4a cells poles during cell-cycle progression (4, 5). However, other aspects containing the bait plasmid. The mixture then was plated on rich of DNA replication still remain unclear. For instance, it is not medium and incubated for 5 h at 30°C. Cells were collected, known how the replication machinery coordinates its action with washed, spread on 120 SC-LUH (synthetic complete medium other cellular processes in a variety of environmental conditions lacking leucine, uracil, and histidine) plates containing an ap- or what the determinants that specify replisome or origin propriate concentration of 3-aminotriazole, and incubated positions within the cell are. Mutants affected in these biological 10–12 days at 30°C. Mating efficiencies were such that 15–30% processes have not been reported yet, possibly because they of the library-containing cells became diploids, allowing us to display weak or inconsistent phenotypes caused by redundant test over 3 ϫ 107 possible interactions in one screen. Hisϩ functions. colonies were transferred on SC-LUA (synthetic complete me- To gain insight into this unexplored area, we used genome- dium lacking leucine, uracil, and adenine) plates and incubated wide yeast two-hybrid screens (6) to identify the proteins that 3–5 days at 30°C. All the Hisϩ Adeϩ colonies were organized in physically associate with known replication proteins from the 96-well plates and subjected to PCR amplification. Inserts Gram-positive bacterium Bacillus subtilis. To circumvent one of junctions with the GAL4 activation domain (AD) were se- the main limitations of the approach, the false-positive interac- quenced and compared with the B. subtilis genome by using the tions, we verified experimentally the specificity of every potential BLAST program and the MICADO database. interaction identified in the screens. The resulting protein net- work is composed of 91 specific interactions connecting 69 Interaction Specificity. To screen out false-positive interactions, proteins. Over one fourth of the interactions were described protein-encoding prey plasmids were rescued from Hisϩ Adeϩ previously in bacteria or eukaryotes, showing that our approach colonies in E. coli KC8 strain, purified, and reintroduced in yields biologically significant interactions. The remaining inter- PJ69–4␣ strain by transformation. For each prey, several trans- actions are previously uncharacterized, and in combination with formants mated with bait-containing PJ69–4a strains as de- data from the literature, many of their biological roles can be scribed in ref. 14. Cells were transferred to SC-LU medium with hypothesized. They link DNA replication with DNA recombi- a replicating tool and incubated 2–3 days at 30°C to select for nation and repair, potential origin- and replisome-anchoring diploids, which then were transferred to SC-LUA͞SC-LUH membrane complexes, signaling pathways, and numerous pro- supplemented with 0.5 mM 3-aminotriazole and SC-LU plates. teins of unknown function. Moreover, we show that the unknown protein YabA, similar only to unknown proteins, acts in initia- tion control, as predicted from protein interactions. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: AD, activation domain; BD, GAL4 DNA binding domain; Pol, DNA polymer- Materials and Methods ase; BSL, B. subtilis genomic library; GFP, green fluorescent protein; MCP, methyl-accepting Strains, Plasmids, and Media. B. subtilis ORFs (7) were PCR- chemotaxis protein. amplified from strain 168 genomic DNA and cloned into a ‡To whom reprint requests should be addressed. E-mail: [email protected]. 8342–8347 ͉ PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.122040799 Downloaded by guest on September 27, 2021 Interaction phenotypes were scored after 10–12 days of growth because of the self-activation of the reporter genes induced by at 30°C. the N-terminal part of the protein. For tri-hybrid experiments, PJ69–4a strain was cotransformed The first cycle of library screenings yielded 38 specific protein– by different combinations of bait and 3H vectors. Uraϩ Trpϩ protein interactions. In the second cycle the ORFs correspond- colonies were mated with PJ69–4␣ strains containing various ing to seven preys were used as baits, and in the third cycle two prey vectors, and diploids were selected on SC-LUW (synthetic ORFs were used as baits, adding to a total of 32 new interactions. complete medium lacking leucine, uracil, and tryptophan). In addition to the screenings, most of the bait proteins also were Interaction phenotypes were scored by replica-plating the dip- expressed as preys and used in specificity assays. These assays loids onto selective plates. SC-LUWH (SC-LUW lacking histi- revealed 21 interactions that were not detected in the screens, dine and containing 0.5 mM 3-aminotriazole) and SC-LUWA possibly because of nonsaturated screens or the scarcity of (SC-LUW lacking adenine). full-length ORFs in the prey library. Interestingly, we found that all the preys (46͞46) isolated from the screens as independent ,4؅,6-Diamidino-2-phenylindole Staining and Nucleoid DNA Content overlapping fragments interacted specifically with their bait Analysis. Cells were grown in LB medium to midexponential which is in line with previous observations (21, 22), whereas only ͞ phase (OD600 0.3–0.5) and fixed with glutaraldehyde as de- one prey out of six (24 148) isolated as a unique fragment did scribed previously (15). Fixed samples were examined by phase- so. Although this proportion depends on our experimental contrast and fluorescence microscopy on a Zeiss Axioplan system, it confirms that the level of false positives in genome- equipped with a Plan NEOFLUAR ϫ100͞1.30 objective and a wide two-hybrid screens can be very high (23) and indicates that Princeton Instruments Micromax 1300Y͞HS charge-coupled false interactions have to be eliminated to assess correctly the device camera (Roper Scientific, Trenton, NJ). For DNA- biological significance of the information derived from such content determination, an internal standard was added to each screens. sample and used to determine relative DNA content in each field The resulting network is composed of 91 specific interactions of cells essentially as described in ref. 16. The digital images were involving 69 proteins (Fig. 1). These proteins can be grouped in quantitated by using METAMORPH 4.6 software, and the data were eight functional categories (Table 2), thus placing the DNA- processed with Microsoft EXCEL. The relative DNA contents replication process into a larger biological context.
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