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 network 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 initiation 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. The biolog- given represent an underestimation of the actual DNA content ical significance of these interactions is discussed below. for reasons outlined in ref. 17 and should be multiplied by a

factor of Ϸ1.3 to arrive at actual DNA contents in chromosome Protein-Interacting Domains. Our experiments yielded information MICROBIOLOGY equivalents. on the protein domains required for the interaction with the baits. Isolation of multiple independent overlapping fragments OriC Region Copy-Number Determination. To visualize the location of a prey allowed delimitation of the interaction domain. The of the replication origin, the oriC-labeling system based on the complete data are available in a dedicated database at http:͞͞ lacO array (18) or on the Spo0J-green fluorescent protein (GFP) www-mig.versailles.inra.fr͞bdsi͞SPiD (24). Users can search and fusion (19) were introduced into the wild-type B. subtilis strain visualize the interactions and the interacting domains, as well as (168) and the yabA null mutant (168⌬yabA::phleo). The lacO access functional and bibliography information through links array was amplified and the strains were grown for microscopy with specialized databases. essentially as described in ref. 18. The cells carrying GFP fusions were viewed on agarose-coated slides essentially as described in Deletion of Unknown Genes. Within the network, we have identi- ref. 20. The LacI-GFP strains were grown in minimal medium fied 25 proteins of unknown function (Table 2) having either no (TSM) at 30°C, and the Spo0J-GFP strains were grown on significant sequence similarity to other known proteins (8) or nutrient agar overnight at 30°C. similarity to one or more hypothetical proteins (17). We deleted 10 genes by individually replacing them with a phleomycin Results resistance marker (yabA, yhaM, yjcK, ykaA, ykcC, ykjA, yncB, ytjP, Construction of the Protein Interaction Map. A BSL was constructed ywhK, and yxeE), and disruption mutants were described for 12 in a GAL4 AD (prey) vector in E. coli. It contained over 107 other genes (yacL, yclM, ydeL, yerB, yhcQ, yisN, yklV, ymaF, ytdP, clones with an average of one fusion endpoint every 52 bp along yufO, yxaD, and yxaL)inthetwoB. subtilis functional databases the chromosome. BSL was transferred into yeast and screened MICADO (http:͞͞locus.jouy.inra.fr͞micado) and JAFAN (http:͞͞ with BD hybrid proteins (baits). The screening was performed bacillus.genome.ad.jp). A further three genes (yomI, yoqX, and by mating, generating large numbers of diploids that were plated yqaH) belonged to dispensable integrated prophages. Thus, all directly on selective medium. The preys from all the colonies these genes are individually dispensable for cell survival. In exhibiting the interaction phenotypes were identified by se- contrast, we found that yqeN encoded a protein essential for B. quencing. A systematic verification of the specificity of each subtilis. The YqeN protein showed sequence similarity to the interaction was carried out to eliminate false positives. For this Streptococcus pyogenes clamp-loader subunit HolA (25) and purpose, prey plasmids were isolated from candidate colonies interacted with another clamp-loader subunit, DnaX, and the ␤ and reintroduced in haploid yeast. These prey cells were mated clamp, DnaN (Fig. 1), which is consistent with HolA function. with cells containing (i) the initial bait used to screen the library, Thus, YqeN was renamed HolA. (ii) an empty bait vector, and (iii) at least six unrelated baits, and the resulting diploids were tested for interaction phenotypes. Biologically Relevant Multiprotein Complexes. To test the rele- Interactions were considered specific if reproduced twice inde- vance of the interactions detected, we examined in more detail pendently with the initial bait and not associated with self- the properties of the previously unknown protein YabA, which activation (tested with the empty bait vector) or stickiness (tested is well conserved in Gram-positive bacteria but has no simi- with unrelated baits) of the prey protein. larity with proteins of known function (data not shown). YabA The 13 proteins chosen as initial baits (Table 1) are key appeared to interact with both the initiator protein DnaA and components of the replication apparatus (1). Generally, the the sliding-clamp DnaN (Fig. 1), potentially linking the initi- full-length proteins were fused to the BD. For the large DNA ation and elongation steps of chromosomal replication. In E. polymerases (Pols), PolC and DnaE, fusions with two overlap- coli, a mechanism of initiation control, mediated by the Hda ping protein fragments were constructed. For DnaC, only the protein, acts by inhibiting the DnaA initiator activity through C-terminal fragment of the DNA helicase was used as a bait interaction with DnaN (26, 27). YabA could accomplish a

Noirot-Gros et al. PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 8343 Downloaded by guest on September 27, 2021 Table 1. Features of two-hybrid screens Diploids Hisϩ Adeϩ Pairwise Screen Bait Bait identiﬁcation (ϫ107) colonies interactions

First cycle DnaB Primosome subunit 11.6 152 4 DnaC_Cter Replicative DNA helicase [151–454] 7.9 451 7 DnaC_Nter Replicative DNA helicase [1–170] s.a. — 0 DnaD Primosome subunit 6.8 82 1 DnaE DNA polymerase [1–1,115] 7.8 48 4 DnaE_Nter DNA polymerase [1–826] 5.1 52 1 DnaE_Cter DNA polymerase [286–1,115] 14.5 8 1 DnaG DNA primase 13.3 51 8 DnaI Primosome subunit 8.8 277 2 DnaN DNA sliding clamp (␤) 4.2 209 6 DnaX Replisome subunit (␶) 6.4 11 3 HolB Replisome subunit (␦Ј) 16.7 20 1 PcrA DNA helicase 7.6 30 4 PolC_Nter DNA polymerase III [1–993] 14.0 11 1 PolC_Cter DNA polymerase III [435–1,438] 12.1 5 1 PriA Primosome subunit 9.1 8 0 HolA Replisome subunit (␦) 9.8 312 6 Second cycle YabA Prey of DnaN 12.3 174 7 YhaM Prey of DnaC 14.8 204 3 YhaN Prey of DnaG 8.5 61 1 YjcK Prey of DnaE 10.4 38 1 YtjP Prey of DnaE 12.2 24 1 YxaL Prey of PcrA 8.8 450 8 YxeE Prey of DnaG s.a. — 0 Third cycle DnaA Prey of YabA 12.6 468 4 TlpA Prey of YabA 9.1 440 16

Baits were GAL4 DNA-binding fusion with full-length proteins except for PolC, DnaC, and DnaE, where fusions with two overlapping protein fragments were constructed. The coordinates of the fragments in amino acids are indicated in brackets. Greek letters indicate polymerase subunit designations in bacteria (25). The lack of correlation between the number of Hisϩ Adeϩ colonies and the number of speciﬁc pairwise interactions is caused in part by the preys isolated as multiple overlapping fragments and to individual fragments obtained several times because of the library’s high redundancy. Also, the speciﬁcity assays revealed additional pairwise interactions that were not detected in the screens. s.a., self-activating bait.

similar type of initiation control by bringing together DnaA increased, even taking into account the increased average cell and DnaN in a multiprotein complex. We showed that an length (Fig. 3 E and G). This increase was particularly apparent interaction between DnaA and DnaN was detected, provided with the Spo0J-GFP (Fig. 3G), possibly because the Spo0J that YabA was coexpressed in the cell (Fig. 2). This result protein associates with sites covering a broader region of the indicates that YabA can act as a bridge between DnaA and chromosome than with the LacO array (29). For the lacI-lacO DnaN. system, which more specifically tags oriC, we determined the We constructed a nonpolar yabA deletion mutation, which was average number of foci per unit cell length; this was 3.46 for the viable but showed a greatly reduced growth rate especially in wild type but almost 40% higher, 5.04, for the mutant cells (93 minimal media (data not shown). Microscopic examination of and 143 foci counted, respectively). Furthermore, an underes- the mutant (Fig. 3B) revealed an abnormal nucleoid distribution, timation of foci number in the yabA mutant is likely because of with increased cell length, which is probably an indirect conse- the greater probability of two foci coinciding and being counted quence of the nucleoid effect. Several lines of evidence suggested as a single focus and because some GFP foci would be out of that this phenotype was associated with increased initiation of focus in the longer and more twisted cells. Taken together, these DNA replication. First, we measured the relative DNA content results indicate that YabA acts as a negative regulator of of nucleoids of the wild-type and mutant strains (Fig. 3C), which initiation at oriC. revealed that the mutant had many nucleoids of much higher relative DNA content (Ͼ2.5) than the wild type, giving a Discussion significantly higher average DNA content of 2.3 versus 1.7 Specific Interactions Are Biologically Relevant. To assess the chromosome equivalents per nucleoid. We then directly exam- biological relevance of our screens, we compared our data with ined the copy number of the oriC region in wild-type and yabA the known interactions between replisome proteins. We found mutant cells by using two marker systems: a lacO͞LacI-GFP that the DnaC helicase (the E. coli DnaB homolog) interacts tagging system (18) or Spo0J-GFP, which is associated naturally with the DnaG primase and DnaX, the ␶ subunit of PolIII with the oriC region (28, 29). Previous work has shown that each holoenzyme. DnaX interacts with HolB and HolA (YqeN) to focus represents one copy of the oriC region of the cell or two form the ␤ clamp loader (25). The replicative polymerase PolC recently replicated copies. In wild-type cells containing either interacts with DnaX and DnaN, the ␤ sliding clamp. Self- GFP protein, discrete foci were located mainly at regularly interactions were observed with DnaN, DnaX, and the DnaC spaced intervals in the cells and cell chains (Fig. 3 D and F). In helicase. All the corresponding interactions in E. coli have both cases, the general appearance was similar to that reported been shown to be required for replication fork function (2). previously, with typically 2–4 foci per cell. In contrast, in the Also, we found that DnaI interacts with the DnaC helicase, as yabA mutant, the general number of foci per cell was clearly do the DnaI homologues from integrated phages, XkdC and

8344 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.122040799 Noirot-Gros et al. Downloaded by guest on September 27, 2021 MICROBIOLOGY

Fig. 1. Protein–protein interaction map. Proteins are symbolized by ovals, and protein names are as described in ref. 7 except for HolA (YqeN). Primary baits are indicated by a double blue line except PriA and DnaD, which were omitted. The dashed line symbolizes a bacterial replisome. The arrows are oriented from the bait to the prey. The complete data set including the interacting domains of proteins can be viewed at http:͞͞www-mig.versailles.inra.fr͞bdsi͞SPiD. Functional categories are represented with colors: orange, DNA replication͞recombination͞repair; dark blue, mobility and chemotaxis; light blue, signal transduction; light green, transcription; pink, protein synthesis; dark green, metabolism of carbohydrates and amino acids; purple, cell division; and yellow, unknown.

YqaM. Recently it was proposed, based on the DnaI–DnaC expected limitation of our approach, inherent to the use of interaction, that DnaI acts as a helicase loader (30). Interest- DNA-replication proteins as baits, is that some interactions ingly, the PolC–DnaX interaction was shown to be relatively might depend on binding to DNA. One such example is that of weak in Gram-positive bacteria (25). Its detection with the B. the primosomal proteins DnaB and DnaD, which we found to subtilis proteins shows that our screens are highly sensitive. An interact with themselves, consistent with the multimerization

Table 2. Summary of experimental results Description Total

Yeast two-hybrid screens B. subtilis proteins used as baits 22 Two-hybrid screens performed 25 Baits yielding specific interactions 20 Specific interactions identified 91 Discrete interacting protein pairs 86 Average interactions per bait 4 Functional classification* Interacting proteins 69 DNA replication͞repair͞recombination 25 Mobility and chemotaxis 5 Signal transduction 3 Transcription 2 Fig. 2. YabA acts as a protein bridge between DnaA and DnaN. DnaA was Protein synthesis, elongation 1 expressed as bait (BD-DnaA), and DnaN was expressed as prey (AD-DnaN). Metabolism of carbohydrates, amino-acids, 7 YabA was expressed as the trihybrid protein (3H-YabA). Negative controls and nucleotides include BD, 3H, and AD expressed from empty vectors. The positive control is Cell division 1 AD-YabA. The different combinations of bait and 3H vectors in PJ69–4a are Unknown 25 indicated at the left of each row. The preys expressed in each strain are indicated at the top of each column. Interaction phenotypes were scored by *As defined in ref. 7. replica-plating the diploids onto selective plates SC-LUWA.

Noirot-Gros et al. PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 8345 Downloaded by guest on September 27, 2021 Pol. DnaE, similar to PolC, is required for the elongation step of chromosome replication, and both enzymes exhibit similar localization patterns (38), which suggests that PolI might play a structural role within the replisome in addition to a role in the processing of Okazaki fragments. The DnaG primase interacted with YirY and YhaN, which are homologs of E. coli SbcC. Moreover, YhaN interacted with YhaO, a SbcD homolog. The E. coli SbcCD nuclease that belongs to the structural mainte- nance of chromosomes (SMC) protein family, is involved in DNA recombination and exhibits an ATP-dependent degrada- tion of DNA hairpin structures (39). The interaction of SbcCD- like enzymes with the DnaG primase supports the hypothesis that these enzymes act on the lagging strand at the replication fork (39). The finding of direct interactions between replication and recombination͞repair proteins provides insights into how these two processes are linked. DNA repair and recombination functions are of critical importance for the restoration of replication forks, which often are arrested during chromosomal replication (for reviews see refs. 40 and 41). In conclusion, our approach allowed a meaningful reconstruction of the B. subtilis replication machinery, strongly indicating that specific interactions have biological relevance.

Replication Is Linked with the Membrane and Signaling Pathways. Interestingly, a complex mesh of interactions linking DNA replication with membrane-associated protein complexes and signaling pathways were detected. The DnaC helicase interacted with BfmBAB, and the DnaG primase interacted with AcoC, BfmBAB, and PdhC. These proteins are components of three Fig. 3. Effects of a yabA mutation on cellular DNA content and initiation of very large (5–10-megadalton) multienzyme complexes that cat- DNA replication in vivo.(A and B) Combined phase-contrast and 4Ј,6- alyze the decarboxylation of acetoin and 2-oxo acids and transfer diamidino-2-phenylindole (DNA) images of wild type (A, WT) and yabA mu- the corresponding acyl groups to CoA (42). PdhC, a subunit of tant (B) strains grown in LB medium. (C) Analysis of the relative DNA content the pyruvate dehydrogenase complex, was identified recently as of cells grown as described for A and B.(D and E) Analysis of oriC copy number a membrane-associated regulator of B. subtilis DNA replication in wild-type (D) and yabA mutant (E) cells expressing a LacI-GFP fusion tar- in vitro (43). Thus, PdhC might be involved in DNA replication geted to a lacO cassette located near oriC.(F and G) Analysis of oriC copy through its direct interaction with DNA primase. Noteworthy is number in wild-type (F) and yabA mutant (G) cells expressing an Spo0J-GFP the possibility that the 2-oxo acid dehydrogenases might transfer fusion. acyl groups to the DnaC and DnaG proteins. Considering the lack of knowledge about the role of protein acylation in pro- observed with the purified proteins (31, 32). However, we did karyotes, this hypothesis cannot be ruled out. not detect the PriA–DnaD–DnaB interactions, which depend Several methyl-accepting chemotaxis proteins (MCPs), which on the presence of a specific DNA substrate (32). Overall, we are transmembrane receptors, seem to be linked to DNA detected 16 of 20 interactions involving replisome and initia- replication. The cytosolic part of TlpA interacts with 17 different protein partners. Interactions with TlpA, McpA, the MCP tion proteins, which were reported in the literature, either in homologs YhfV and YvaQ, and the chemoreceptor-controlled B. subtilis or other model organisms. This proportion suggests kinase CheA were expected based on E. coli data (44). They that the number of false negatives is low in our experiments. support the hypothesis that the different chemoreceptors inter- Thus, with the limitation that all possible interactions could act with each other to form composite complexes. The MCPs not be detected, many biologically relevant interactions in- form large clusters located at the cell poles in E. coli (45) and B. volved in DNA replication have been revealed. subtilis (46). We found that McpA and TlpA interact with YabA, Other known proteins seem to be associated with the DNA- suggesting that YabA might bring into close proximity the MCPs replication machinery (Fig. 1). Besides PolC, DnaN was found to ͞ and the initiator DnaA. Furthermore, the DnaB initiator protein interact with YqjH, a UmuC DinB homolog, and MutL, a (different from the E. coli DnaB helicase) interacts with McpA mismatch repair protein. E. coli DnaN was shown to interact in Ј and YvaQ. DnaB frequently localizes near the cell poles (30). vitro with the translesional Pols UmuD 2C and DinB and in- Thus, our data suggest that the proximity of the polar MCPs with crease their processivity to various extents (33, 34). The role of initiation proteins might play a functional role, possibly at a stage the DnaN–MutL interaction is suggested by the function in DNA of the cell cycle when the origins are also oriented preferentially mismatch repair of the interaction between the yeast sliding toward the poles (47). The interactions of replication proteins clamp, PCNA, and the MutL homolog MLH1 (35). Interestingly, with massive membrane-associated protein complexes (Pdh and the sliding clamp was found to interact with MutS or a MutS MCPs) might be important in determining some of the striking homolog in E. coli and yeast (35, 36), suggesting that interaction localization patterns that have been described (3, 30, 47). with mismatch repair is universally conserved, albeit through MCPs may be involved in yet other aspects of DNA metab- different protein complexes in the different organisms. Remark- olism, because we found that TlpA interacts with GyrA (a ably, B. subtilis MutL and MutS proteins were shown to form foci subunit of gyrase), RecF and YirY (involved in recombinational that can assemble at the replication machinery (37). PolI inter- repair), and DinG (a damage-inducible helicase). TlpA interacts acted with HolB, a subunit of the ␤ clamp loader, suggesting that also with FtsH, an ATP- and Zn2ϩ-dependent metalloprotease as in E. coli, PolI might function with the ␤ clamp (36). Strikingly, involved in cell division (48). In dividing cells, FtsH accumulates PolI interacted with PolIII (PolC) and DnaE, another class C at the septum (48) where the MCPs are located predivisionally

8346 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.122040799 Noirot-Gros et al. Downloaded by guest on September 27, 2021 (46). Perhaps, the FtsH–TlpA interaction might fulfill its func- this result perhaps is not surprising. The proteins might have tion at the time of cell division. Interestingly, the link between redundant functions or play various accessory roles in DNA TlpA and the DegS kinase indicates that in addition to CheA͞ replication. However, they now can be placed into biological CheY, the DegS͞DegU two-component regulatory system (49) context, and testable hypotheses about their functions can be might be related to signal transduction in chemotaxis. generated. To illustrate this point, we tested the function of The interaction network reveals additional links between YabA, a protein of unknown function well conserved in Gram- DNA replication and proteins involved in signaling. HolA positive bacteria. YabA interacted with both DnaA and DnaN, (YqeN) interacts with DnaX, as expected, but also with CitS, a suggesting that it might be involved in initiation control, based membrane-bound sensor kinase (49), and LepA, a GTPase of on the Hda-mediated control mechanism described in E. coli (26, unknown function, universally conserved among bacteria (50). 27). We showed that YabA can act as a protein bridge between CitS is involved in the regulation of magnesium-citrate transport DnaA and DnaN. In the absence of a functional YabA protein, (51). It appears to interact with HolA through a cytosolic domain the average DNA content of nucleoids was much higher, and the flanking the histidine phosphotransferase domain, raising the number of oriC copies per cell unit was increased significantly possibility that the interaction might depend on the phosphor- compared with wild type. Taken together, these results indicate ylation state of CitS. LepA is homologous to protein synthesis that YabA acts as a negative regulator of initiation at oriC.In elongation-factor GTPases (50), suggesting that LepA might act contrast to E. coli Hda, YabA shares no homology with DnaA. to coordinate DNA replication and protein synthesis. Thus, our Moreover, YabA interacts with the MCPs that are linked to initiation at oriC (see above) and with other proteins that also data reveal potential signaling pathways that might act to tune might have a role in initiation control. These findings suggest DNA replication with the global cell physiology. that YabA mediates a complex mechanism of initiation control and open the way to the study of initiation control in Gram- Previously Uncharacterized Proteins Involved in DNA Replication. positive bacteria. Strikingly, 36% of the proteins in our interaction network have no known function. We have shown that none of them individ- We thank B. Michel, P. Polard, M. A. Petit, E. 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