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Journal of Bacteriology, January 2009, p. 333-346, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.00331-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery{triangledown} ,{dagger}

Gouzel Karimova, Carine Robichon, and Daniel Ladant*

Institut Pasteur, CNRS URA 2185, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 25 rue du Dr Roux, Paris Cedex 15, France

Received 5 March 2008/ Accepted 24 October 2008


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ABSTRACT
 
Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.


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INTRODUCTION
 
Cell division is a fundamental process in all organisms. In Escherichia coli, it requires the coordinated constriction of the three layers of the gram-negative cell envelope: the invagination of the inner membrane and the biosynthesis of septal peptidoglycan, accompanied by the invagination of the outer membrane. The inner membrane invagination is mediated by the divisome, an assembly of proteins that forms a ring-like structure at midcell. In E. coli, the divisome includes at least 10 essential proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN) and a set of nonessential proteins (ZapA, ZapB, FtsE, FtsX, FtsP [SufI], AmiC, and EnvC) (19, 34, 45, 55, 56). The cell divisome formation starts with the polymerization of the FtsZ protein at midcell to form the so-called Z ring. This structure marks the future division site and provides a scaffold for the recruitment of the other cell division proteins (3, 7, 43, 52, 53). Numerous studies showed that the divisome proteins are sequentially recruited to the septum in E. coli. First, two FtsZ binding proteins, FtsA and ZipA, almost simultaneously localize at midcell independently of each other, presumably to stabilize the Z-ring structure. The nonessential accessory proteins ZapA and ZapB that are able to directly associate with FtsZ in vivo (27, 28, 32) may also participate in stabilizing the Z ring (51). Then, a series of integral membrane proteins are recruited to the septum in a sequential manner in the following order: FtsK, FtsQ, FtsL/FtsB, FtsW, FtsI, and FtsN (for reviews, see references 34, 45, 54, and 56). The assembly of these proteins with the divisome is essential for the progression and completion of cytokinesis. Several nonessential proteins, AmiC (10), EnvC (9), and a trans-envelope Tol-Pal complex (24) also associate with the cell division machinery to facilitate the invagination of the outer membrane during the division process and the separation of daughter cells. Under certain stress conditions, additional proteins, FtsX, FtsE, and FtsP (SufI) also become essential for the E. coli division process (47, 48, 50).

Despite the fact that much is known about the individual Fts proteins in terms of sequence, membrane topology, and localization interdependency, the precise functions of most of these proteins remain largely unknown. Besides, the molecular basis of their ordered recruitment to midcell is still unclear. Immunoprecipitation studies have demonstrated the existence of a trimeric complex made of FtsQ/FtsL/FtsB that could assemble outside of the division septum independently of the other components (12). Independent studies, using two different bacterial two-hybrid assays, revealed a complex interaction network among many of the Fts proteins (17, 38) that could not be easily reconciled with the ordered recruitment of the Fts proteins to the septum. Recently, Goehring and coauthors used a method called premature targeting to examine the associations among the E. coli cell division proteins (27, 28). They also clearly showed that the linear assembly model is an oversimplified view of the real process.

One current attractive idea is that the assembly of the divisome machinery may result from the sequential association of several preformed subcomplexes, namely, a cytosolic protoring complex (FtsZ/FtsA/ZipA/ZapA/ZapB), an inner membrane intermediate complex (FtsK/FtsQ/FtsL/FtsB), and a membrane/periplasmic peptidoglycan synthesis-separation machinery (FtsW/FtsI/FtsN/PBP1b/AmiC/EnvC/Tol-Pal complex) (1, 54). Yet, how these complexes assemble during the cell division process remains to be established.

In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. For this purpose, we used a bacterial two-hybrid approach (bacterial adenylate cyclase two hybrid [BACTH]) that is based on the interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade in E. coli (39). We have previously successfully applied this BACTH system to analyze interactions among Fts proteins (38). In this work, we performed library screens using as bait FtsL, a 13-kDa bitopic membrane protein, whose periplasmic domain has a repeated heptad motif characteristic of leucine zippers that could be involved in interactions with other E. coli divisome components (25, 26, 33). FtsL is known to form a trimeric complex together with FtsQ and FtsB (12), but its precise function is still unknown. We have isolated, as an interacting partner of FtsL, a 72-residue integral membrane protein YmgF, of unknown function. YmgF was found to associate with many E. coli cell division proteins in addition to FtsL and was shown to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. In addition, when overproduced, YmgF could partially overcome the thermosensitivity of the ftsQ1(Ts) mutant and restore the ftsQ1 viability under low-osmolarity growth conditions. Taken together, these data suggest that YmgF, although previously shown to be not essential for cell viability (6, 23, 31), might be a novel component of the E. coli cell division machinery.


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MATERIALS AND METHODS
 
General methods. Bacteria were routinely grown at 30°C in Luria-Bertani (LB) broth (0.5% yeast extract, 1% tryptone, 0.5% NaCl) (46). When necessary, LB broth with no NaCl added (LB0) and LB broth containing 1% NaCl (LB1) were used. Unless stated otherwise, antibiotics were added at the following concentrations: kanamycin (50 µg/ml), ampicillin (100 µg/ml), tetracycline (30 µg/ml), and chloramphenicol (30 µg/ml). Transductions with P1 bacteriophage were performed essentially as described previously (46). Standard protocols for molecular cloning, PCR, DNA analysis, and transformation were used (49). PCR was performed with DyNAzyme EXT polymerase from Finnzymes (Espoo, Finland). Oligonucleotides were from MWG Biotech (Ebersberg, Germany). Unless otherwise indicated, genomic DNA from E. coli K-12 MG1665 was used as a template in the PCR procedures. DNA sequencing was carried out by the company Genome Express (Meylan, France).

Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are described in Table 1 and Table 2, respectively. The E. coli strain XL1-Blue (Stratagene) was used in all of the routine cloning experiments. E. coli DH5{alpha} (laboratory collection) was used as a host to study membrane protein topology. E. coli NM554 (laboratory collection) was utilized as a host for promoter probing studies. BACTH complementation assays and BACTH library screening were carried out in the E. coli cya strain DHM1 (38). MG1655 (wild-type E. coli strain) and AK2000 (a cya derivative of MG1655 [see below]) were used as sources of DNA for the construction of BACTH DNA libraries, which were propagated in strain DH10B (Invitrogen).


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  		TABLE 1. Bacterial strains used in this study


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  		TABLE 2. Plasmids used in this study

Strain AK2000, a cya derivative of E. coli MG1655 was constructed as follows. First, the cya-854 allele (carrying a 200-bp deletion within the cya gene) was introduced into MG1655 together with a Tn10 insertion in the nearby ilv locus using P1 lysate made on strain SA2755 (laboratory collection). To accomplish that, tetracycline-resistant (Tcr) P1 transductants were plated on MacConkey agar plates containing 1% maltose (46), and white colonies, which are unable to ferment this sugar (i.e., Mal– Cya–) were isolated. One clone (named AK1999) was used for further studies. Next, the wild-type ilv locus from strain MG1655 was transduced into AK1999 by selecting transductants that could grow on minimal M63 agar containing glucose (46). Several clones were reisolated on MacConkey agar with maltose, and one ilv+ clone that had retained the cya allele (i.e., exhibiting a Mal– phenotype) was kept as AK2000.

Strain MG2005 is a derivative of MG1655, in which the full-length ymgF open reading frame (ORF) has been deleted. This strain was constructed by P1 transduction of the {Delta}ymgF::kan (the recombinant gene encoding the ymgF ORF replaced by the kanamycin cassette) locus from strain JW1156 (6) into MG1655 by selecting for kanamycin-resistant (Kmr) colonies.

MG2006 is a merodiploid strain that contains a translational fusion ymgF-gfp at the lambda attachment site (attB) in addition to a wild-type copy of the ymgF gene in its normal chromosomal locus. It was created by integration of plasmid pInCh-ymgF into the chromosome of MG1655 using the {lambda}InCh procedure (11). Plasmid pInCh-ymgF was constructed by subcloning into pTrc99A, an ApaI-HindIII fragment from plasmid pCA24N-ymgF that encodes a part of lacIq and an ymgF-gfp fusion under the control of an isopropyl-β-D-galactopyranoside (IPTG)-inducible T5/lac promoter (42).

Strain MG2007 is a derivative of MG2006, in which the full-length ymgF ORF has been deleted. This strain was constructed as for MG2005 by P1 transduction of the {Delta}ymgF::kan locus from strain JW1156 (6) into MG2006 by selecting for kanamycin-resistant (Kmr) colonies.

Strains MG06Ats, MG06Qts, and MG06Zts are derivatives of MG2006 that harbor thermosensitive alleles of ftsA, ftsQ, and ftsZ, respectively. They were constructed by P1 transduction (selecting for Tcr clones) of the corresponding locus from strains MCA12, MCQ1, and MCZ84, respectively, into MG2006. To confirm cotransduction of the thermosensitive fts alleles, the resulting Tcr transductants were visually screened for their ability to form filamentous cells after the growth temperature was shifted from 30°C to 42°C.

Strains MG06AmiC, MG06EnvC, MG06TolA, MG06TolQ, and MG06Pal are derivatives of MG2006 that contain null alleles of amiC ({Delta}amiC::kan), envC ({Delta}envC::kan), tolA ({Delta}tolA::kan), tolQ ({Delta}tolQ::kan), and pal ({Delta}pal::kan), respectively. These strains were constructed by P1 transduction of the corresponding mutant alleles from the Keio collection strains (Table 1) into MG2006.

Strains MG06Qnull and MG06Nnull are derivatives of MG2006 that contain a null allele of ftsQ and ftsN, respectively. To construct MG06Qnull, MG2006 was first transformed with pJC10, a chloramphenicol-resistant plasmid that carries a wild-type copy of ftsQ under the control of an arabinose-dependent PBAD promoter (14). The ftsQE14::kan locus from strain JOE417 was then P1 transduced into E. coli MG2006(pJC10) by selecting for kanamycin-resistant (and chloramphenicol-resistant) transductants on LB agar supplemented with the corresponding antibiotics and arabinose (0.2%) to induce expression of the complementing ftsQ allele from the plasmid. Several clones were selected and grown in M63 medium plus antibiotics and in the presence of 0.2% glucose (to repress expression from the PBAD promoter) to test for FtsQ depletion, as visualized by cell filamentation. One clone was chosen and named MG06Qnull.

A similar strategy was applied to construct the MG06Nnull strain by using plasmid pJC8 and a P1 lysate made on strain JOE565 (13).

To create strain MG2008, first, the plasmid pBAD33-ymgF, expressing YmgF under the control of an arabinose-inducible promoter was constructed. To accomplish that, the ymg structural part containing the putative Shine-Dalgarno region was amplified from MG1655 chromosomal DNA using oligonucleotides ymgFSD and ymgF72 (Table 3). The amplified DNA fragment was digested with SacI and XbaI (the corresponding restriction sites were included in the oligonucleotide sequences) and inserted into pBAD33 linearized with the same enzymes. After transformation of strain JOE309 with the resulting plasmid pBAD33-ymgF (13), these cells of JOE309(pBAD33-ymgF) were used as a recipient for P1 transduction of the {Delta}ymgF::kan locus from strain JW1156 (6) by selecting for kanamycin-resistant (Kmr) colonies on LB agar plates supplemented with 0.2% arabinose. For a control, the {Delta}ymgF::kan locus was also P1 transduced into JOE309 strain transformed with the empty vector pBAD33, thus creating MG2009.


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  		TABLE 3. Oligonucleotides used in this study

E. coli BACTH library construction and screening. A library of E. coli polypeptides fused to the C terminus of the Bordetella pertussis adenylate cyclase T18 fragment was constructed using vector pUT18BC. This vector is a derivative of pUT18C (36) modified by insertion of the hybridized oligonucleotides BstX1 and BstX2, which harbor two nonsymmetrical BstXI recognition sites (Table 3), between the KpnI and ClaI sites of pUT18C. Genomic DNA from E. coli K-12 strain MG1665 was prepared from a 100-ml overnight culture essentially by the method of Dale and Greenaway (16) by sodium dodecyl sulfate (SDS)-mediated cell lysis, proteinase K treatment, and phenol extraction. About 50 µg of the purified genomic DNA was randomly sheared by sonication (to yield fragments ranging from 300 to 1,000 bp) and end repaired with mung bean nuclease and a mixture of T4 DNA polymerase, Klenow fragment, and deoxynucleoside triphosphate. The blunt-ended DNA fragments were then ligated with annealed adapter oligonucleotides BstX3 and BstX4 (Table 3) for 16 to 19 h at 16°C. After removal of the excess adapters, the DNA fragments were ligated to the pUT18BC vector digested with BstXI. The ligation mixture was used to transform electrocompetent DH10B (ElectroMAX; Invitrogen) cells, giving rise to approximately 5 x 105 to 8 x 105 independent clones. PCR amplification of inserted fragments from 20 randomly chosen clones indicated a mean size distribution of the inserts ranging from 200 to 1,500 bp. All the independent colonies were then pooled; the plasmid DNA was purified from the mixture and used as a stock BACTH library.

From the initial screens carried out with this BACTH library, it turned out that many clones that were isolated as Mal+ on the selective medium (see below) harbored plasmids that carried a fragment of the endogenous E. coli cya gene. To avoid the problem of a high frequency of false-positive clones, a new genomic library was constructed as described above using genomic DNA purified from strain AK2000, a MG1655 derivative that carries a deletion within the cya gene.

For library screen experiments, the E. coli cell division protein FtsL was chosen as bait. Plasmid pKT25-ftsL that encodes T25-FtsL hybrid protein was first introduced into strain DHM1. The resultant strain DHM1(pKT25-ftsL) was made electrocompetent and transformed with the E. coli DNA library using 50 to 100 ng of DNA. After incubation in rich LB medium at 30°C for 90 min, cells were collected by centrifugation, washed several times with M63 medium, and plated (approximately 5 x 105 transformants/plate) on M63 minimal agar supplemented with maltose (0.2%), 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) (40 µg/ml), IPTG (0.5 mM), kanamycin (25 µg/ml), and ampicillin (50 µg/ml). Plates were incubated at 30°C for 5 to 10 days until the appearance of blue Cya+ (Mal+ and Lac+) colonies that were reisolated and further characterized by sequencing of the DNA inserts.

ymgF promoter analysis. Plasmid pYmgF-lac is a derivative of plasmid pTL61T that encodes a promoterless lacZ gene (44). A 96-bp DNA fragment containing the putative promoter region and the first two codons of ymgF were PCR amplified (with the primers ymgF3 and ymgF4) and subcloned between the EcoRI and SalI sites of pTL61T.

To probe promoter activity, strain NM554 was transformed with plasmid pYmgF-lac, and the transformants were plated on LB agar containing X-Gal. To measure β-galactosidase activity, the bacteria were grown overnight at 37°C in LB broth with ampicillin (100 µg/ml), permeabilized, and assayed as described previously (38).

Membrane topology analysis. To study the membrane topology of YmgF, we first constructed plasmid pKTop that encodes a dual pho-lac reporter as described previously (4). The chimeric pho-lac DNA fragment was constructed by a PCR-mediated overlap extension technique (36). To do that, in the first step, two DNA fragments, encoding a part of the alkaline phosphatase PhoA (amino acids [aa] 22 to 472) and β-galactosidase LacZ (aa 4 to 60) were PCR amplified from the E. coli MG1655 genomic DNA using oligonucleotides phoA1 and phoA2 and oligonucleotides lac1 and lac2, respectively (Table 3). Fifteen nucleotides from the lacZ DNA were introduced into the reverse phoA2 primer to generate an overlapping region between these two PCR products. The purified pho and lac DNA fragments were used as the template in a second PCR to create a fused pho-lac DNA that was amplified with the external primers (phoA1 and lac2). After digestion with SacI and ApaLI restriction enzymes, this resulting pho-lac DNA fragment was ligated between the corresponding sites of plasmid pKNT25 (38) to create pKTop (the pho-lac fusion replacing the T25 ORF). A set of pKTop derivatives were then constructed. In these plasmids, the dual pho-lac reporter was fused in frame after selected ymgF codons: D8 (pKTop-YmgF1-8), M21 (pKTop-YmgF1-21), K32 (pKTop-YmgF1-32), E39 (pKTop-YmgF1-39), N48 (pKTop-YmgF1-48), L57 (pKTop-YmgF1-57), and Q72 (pKTop-YmgF1-72). The corresponding ymgF fragments were PCR amplified from genomic DNA from strain MG1665 by using appropriate primers (Table 3) and subcloned between the BamHI and KpnI sites of pKTop. In the case of pKTop-YmgF1-8, the two complementary oligonucleotides ymgF8d and ymgF8r (designed to code for the first eight residues of YmgF) were hybridized and directly ligated between the BamHI and KpnI sites of pKTop.

For membrane protein topology assays in vivo, E. coli DH5{alpha} was transformed with the resulting plasmids and plated on dual-indicator plates containing LB agar with 5-bromo-4-chloro-3-indolyl phosphate disodium salt (X-Phos) (Sigma) at a concentration of 80 µg/ml and 6-chloro-3-indolyl-β-D-galactoside (Red-Gal) (Sigma) at a concentration of 100 µg/ml as indicators and IPTG (1 mM), 50 mM phosphate buffer (pH 7.0), and 50 µg/ml kanamycin.

Plate viability spot assays. To construct plasmid pUC19-ymgF, used in complementation assays of the thermosensitive fts strains, a 347-bp DNA fragment containing the putative promoter region and the full-length ORF of ymgF was PCR amplified from the MG1655 genome using oligonucleotides ymgF5 and ymgF6 (Table 3). The purified PCR fragment was subcloned between the BamHI and KpnI sites of pUC19. The following E. coli strains harboring various fts temperature-sensitive alleles were transformed with the resulting plasmid: MCZ84 with ftsZ(Ts), MCA12 with ftsA(Ts), MCI23 with ftsI(Ts), and MCQ1 with ftsQ(Ts). For controls, cells were transformed with an empty pUC19 vector. The viability of the transformants was determined by plate viability spot assays. For plate viability spot assays, the cells were grown overnight in LB broth containing 100 µg/ml ampicillin and 0.2% glucose at 30°C. The next morning, the overnight cultures were diluted 1:200 in fresh LB with ampicillin and glucose and incubated for 3 to 4 h at 30°C. The bacteria were then adjusted to equivalent optical densities at 600 nm, and 10-µl aliquots of serial dilutions (100, 10–1, 10–2, 10–3, 10–4, 10–5, and 10–6) were spotted on LB agar plates supplemented with ampicillin. To examine thermosensitive phenotypes of fts(Ts) mutant strains, the plates were incubated at 30°C or at 42°C for 24 to 32 h. To test the ability to grow under low-salt conditions, the transformants were spotted similarly on either LB0 or LB1 agar (plus ampicillin) and incubated at 30°C for 24 h.

BACTH assays. Plasmids pKT25, pKNT25, pUT18C, pUT18, pKT25-ftsA, pKT25-ftsI, pKT25-ftsL, pKT25-ftsB, pKT25-ftsQ, pKT25-ftsN, pKT25-ftsW, pKT25-ftsX, pKNT25-ftsZ, pKT25-ymgF, pUT18C-ymgF, and pKT25-malG used in the BACTH complementation assays were described previously (38, 40). The pKNT25-ymgF and pUT18-ymgF plasmids that code for the YmgF polypeptide fused in frame to the N terminus of T25 or T18, respectively, were constructed by subcloning a 216-bp DNA fragment encompassing the full-length ymgF ORF (without the stop codon; amplified by PCR with primers ymgF1 and ymgF2) between the BamHI and Acc65I sites of pKNT25 and pUT18. The pKT25-ftsL(FLL) and pKT25-ftsL(LFL) plasmids encode T25-FtsL variants in which, either the cytosolic domain (FLL) or the transmembrane segment (LFL) of FtsL were replaced with a cytosolic or transmembrane segment of the MalF protein (33). To construct these plasmids, the FLL and LFL genes were PCR amplified from plasmid pLD63 or pLD94 (33) using primers F1 and L2 or primersL1 and L2, respectively (Table 3), digested with BamHI and Acc65I, and cloned into pKT25 linearized with the corresponding enzymes.

In the BACTH complementation assays, screening for the ability to metabolize sugar was performed on LB agar plates supplemented with X-Gal, (40 µg/ml), IPTG, (0.5 mM), ampicillin (100 µg/ml), and kanamycin (50 µg/ml). β-Galactosidase activities were assayed by plating DHM1 cells encoding the various combinations of T25 and T18 fusion proteins onto LB medium containing X-Gal and IPTG and incubating the cells at 30°C for 3 to 4 days. For each combination, 40 to 50 randomly chosen clones were resuspended in 500 µl of 0.85% NaCl. Enzymatic assays were then carried out as described previously (38).

Microscopy analysis. Subcellular localization of YmgF was examined by fluorescence microscopy of cells expressing an ymgF-gfp fusion inserted as a single copy into the chromosome.

Microscopy studies to visualize the subcellular localization of YmgF-green fluorescent protein (GFP) fusions were performed on live cells. To do this, the E. coli strains to be examined were grown overnight in LB broth supplemented with 0.2% glucose and appropriate antibiotics at 30°C. The cultures were then diluted 1:200 in minimal M63 medium containing IPTG (100 µM), Casamino Acids, appropriate antibiotics, and glucose and grown to early exponential phase for 3 to 4 h at 30°C. A drop of culture was then deposited on a glass slide and covered with a microslide without any fixation step. The cells were visualized by fluorescence microscopy with a Nikon epifluorescence microscope Eclipse 80i containing a 100x Plan-Apo oil immersion objective and a 100 W mercury lamp. Images were captured with a five-megapixel color charge-coupled-device DS-5Mc camera and ACT-2U software. Images were further processed using Adobe Photoshop software.

For depletion experiments utilizing the thermosensitive alleles of ftsA and ftsZ, the corresponding strains MG06Ats, MG06Zts, MG07Ats, and MG07Zts were grown overnight in LB supplemented with 0.2% glucose and appropriate antibiotics at 30°C and diluted 1:200 in M63 minimal medium and grown to early exponential phase as described above. Bacteria were then diluted 1:2 into fresh medium prewarmed at 42°C; growth was continued for 90 to 120 min until cell filamentation became visible.

For FtsQ depletion experiments using the conditional PBAD promoter, the MG06Qnull strain was grown overnight in LB supplemented with arabinose (0.2%), ampicillin (25 µg/ml), chloramphenicol (30 µg/ml), and kanamycin (50 µg/ml) at 30°C. Cells were then diluted 1:200 into M63 medium containing 100 µM IPTG, 0.2% glucose, and appropriate antibiotics and grown for 3 to 5 h until cell filamentation occurred. For FtsN depletion experiments, the MG06Nnull cells were treated similarly, except that no arabinose was added in the LB medium during the overnight culture.

To visualize the effect of YmgF overproduction on the E. coli cells harboring the temperature-sensitive alleles of ftsZ, ftsA, ftsI, and ftsQ, overnight cultures of the corresponding strains transformed with pUC19 or pUC19-ymgF were diluted 1:200 in LB (plus ampicillin) and incubated to early exponential phase at 30°C. Bacteria were then diluted 1:2 with fresh prewarmed LB (plus ampicillin) and incubated at 42°C for 1 to 2 h. For microscopic observation of the phenotype under low-salt conditions, the cells were grown overnight at 30°C in LB0 or LB1 broth (plus ampicillin). Phase-contrast microscopy of live cells was performed as described above.

Cell fractionation and protein detection. For cell fractionation, overnight cultures of E. coli MG2007 and MG1655 were diluted 1:50 in 100 ml of minimal M63 medium supplemented with glucose and the appropriate antibiotics and incubated 3 h at 30°C. IPTG (50 µM) was then added to induce ymgF-gfp, and cells were incubated for another 3 h at 30°C (optical density at 600 nm of around 0.4). The cells were harvested by centrifugation at 4,000 x g for 10 min at 4°C and resuspended in 2.5 ml of buffer I (10 mM Tris-HCl, 150 mM NaCl [pH 8.0]) supplemented with protease inhibitor cocktail (Roche Pharmaceuticals). Cells were disrupted by sonication and centrifuged for 10 min at 4,000 x g to eliminate unbroken cells and debris. The supernatants (500 µl) (corresponding to the lysates or the total extracts) were precipitated with 10% trichloroacetic acid and resuspended in 50 µl SDS sample buffer (49). The remaining supernatants (2 ml) were centrifuged at 100,000 x g in a Beckman TL100 centrifuge for 1 h at 4°C to collect the membrane fraction. The resulting supernatants (2 ml) corresponding to the soluble fractions were stored at –20°C. The pellets were resuspended in 500 µl of buffer I and solubilized by the addition of 50 µl of 10% n-dodecyl-β-D-maltopyranoside (DDM) (Sigma) and incubation on ice for 1 h. The solubilized membrane proteins were then separated from insoluble material by ultracentrifugation at 100,000 x g for 30 min at 4°C. Insoluble fraction pellets were resuspended in 500 µl of buffer I. The obtained DDM-solubilized membrane protein fractions and insoluble fractions were prepared in 3x SDS sample buffer (49). In parallel, 500-µl samples of the stored soluble fractions were precipitated with 10% trichloroacetic acid and resuspended in 50-µl portions of 1x SDS sample buffer (49). Finally, comparable amounts of each fraction (soluble, insoluble, solubilized membrane, and total extracts) were incubated at 100°C for 5 min and separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were then electrotransferred onto a polyvinylidene difluoride membrane (Millipore) and incubated with an anti-GFP monoclonal antibody (Santa Cruz Biotechnology). After the membrane was washed, it was incubated with horseradish peroxidase-conjugated mouse secondary antiserum (Amersham Biosciences). Bound horseradish peroxidase-labeled antibodies were detected by enhanced chemiluminescence (ECL-Plus kit; Amersham Biosciences).


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RESULTS
 
Two-hybrid screen of a putative interacting partner(s) of FtsL. To identify a putative novel component(s) of the E. coli cell division machinery, we carried out a bacterial two-hybrid (BACTH) screen using FtsL as bait protein. In the BACTH system, the proteins of interest are genetically fused to two complementary fragments (T25 and T18) from the catalytic domain of B. pertussis adenylate cyclase and coexpressed in an E. coli strain deficient in its endogenous adenylate cyclase. Upon interaction of the hybrid proteins, the T25 and T18 fragments can complement each other, leading to cAMP synthesis and in turn to transcriptional activation of catabolic operons (such as the lactose operon or the maltose regulon) (39).

As the first step, we constructed a library of E. coli polypeptides fused to the C terminus of the B. pertussis adenylate cyclase T18 fragment (see Materials and Methods). Briefly, genomic DNA from E. coli MG1655 was randomly fragmented by sonication, ligated with adapters, and cloned into the BACTH vector pUT18BC. After E. coli DH10B cells were transformed, about 5 x 105 to 8 x 105 independent clones were obtained. The plasmid DNA extracted from the pooled colonies was used as a stock BACTH DNA library. This library was then used to transform E. coli DHM1 cells harboring plasmid pKT25-ftsL that expresses a T25-FtsL hybrid protein (38). The transformed cells were plated on minimal medium supplemented with maltose as the sole carbon source, kanamycin, ampicillin, IPTG and X-Gal (to facilitate the detection of Cya-positive clones that are Mal+ and also Lac+): in principle, only cya+ bacteria can grow on this selective medium, as the expression of the maltose regulon is critically dependent upon a functional cAMP-catabolite gene activator protein complex. The resulting cya+ clones were further characterized by DNA sequencing of fragments inserted in the cognate pUT18BC plasmids.

The first screen of this E. coli MG1665 genomic library, using FtsL as bait, identified about 100 Cya+ clones out of a total of ~2 x 106 spread transformants. Twenty of these cya+clones were further analyzed by DNA sequencing of the corresponding pUT18BC inserts. Four clones were found to harbor the same DNA fragment, encoding aa 19 to 61 of the E. coli cell division protein FtsI, fused in frame to T18. This FtsI segment encompasses the transmembrane domain (aa 20 to 43) and a short extension of 18 aa from the periplasmic part of the protein. This is in good agreement with the results of Wissel et al. who showed that a fragment of FtsI encompassing residues 22 to 47 is able to direct GFP to the E. coli septum (57). The other 16 cya+ clones were found to carry various fragments from the endogenous E. coli cya gene: the corresponding plasmids were shown to complement the cya phenotype of DHM1 independently of the plasmid pKT25-FtsL (or pKT25).

To eliminate such false-positive clones, we constructed a second library using the genomic DNA, which was isolated from an E. coli cya strain, AK2000. This new library, which contained about 5 x 105 independent clones, was screened by transformation of E. coli DHM1(pKT25-ftsL), and plating on the same selective medium as described above. In this second screen, 25 cya+ clones were isolated from ~5 x 106 plated transformants, and 10 of these transformants were characterized by DNA sequencing of the inserted fragments. Seven out of these 10 clones contained a fragment from an uncharacterized open reading frame (ymgF gene) that codes for a putative 72-residue integral membrane polypeptide. In all seven clones, the YmgF ORF (either residues 2 to 72 or residues 6 to 72) was fused in frame with the T18 fragment.

We attempted to further characterize this putative inner membrane-associated protein and, particularly, to examine its potential association with E. coli cell division proteins, although prior reports indicated that the ymgF gene is not essential and could be inactivated without affecting the cell viability (6, 23, 31).

YmgF association with the E. coli cell division machinery. To characterize the putative associations of YmgF with the cell division machinery, the full-length protein was tested in BACTH complementation assays with several E. coli Fts proteins, i.e., FtsA, FtsB, FtsI, FtsN, FtsQ, FtsX, FtsW, FtsZ, in addition to FtsL. In the first set of experiments, the ymgF coding region was PCR amplified and cloned into the pUT18C vector to generate recombinant plasmid expressing the YmgF polypeptide fused to the C terminus of the T18 moiety (hybrid protein T18-YmgF). The pUT18C-ymgF plasmid was then used with different pKT25-fts plasmids encoding Fts proteins fused to the C terminus of the T25 fragment (except in the case of FtsZ, which was functional only when fused to the N terminus of T25) to transform bacterial cells (38). Interaction efficiencies were quantified by measuring β-galactosidase activities in E. coli DHM1 cells coexpressing the different pairs of hybrid proteins. These experiments showed that, in addition to FtsL, YmgF was able to efficiently interact in vivo with two other E. coli cell division proteins, FtsI and FtsQ (Fig. 1a).


Figure 1
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  		FIG. 1. BACTH analysis of YmgF interactions with E. coli cell division proteins. The efficiencies of functional complementation between the indicated hybrid proteins were quantified by measuring β-galactosidase activities in E. coli DHM1 cells harboring the corresponding plasmids as described in Materials and Methods. (a) Coexpression of T18-YmgF with T25-Fts proteins; (b) Coexpression of YmgF-T18 with T25-Fts proteins. Coexpression of YmgF-T18 and T25-FtsQ was toxic for the cells, and the level of β-galactosidase activity could not be measured.

In the second set of experiments, YmgF was fused to the T18 fragment via its C terminus rather than its N terminus as described above. The hybrid protein YmgF-T18 was then tested in BACTH complementation assays with the same set of Fts proteins. As shown in Fig. 1b, in this configuration, the interaction network of YmgF was greatly expanded: the protein was able to dimerize and appeared to associate with essentially all the tested E. coli cell division proteins (FtsA, FtsB, FtsI, FtsL, FtsN, FtsW, FtsX, and FtsZ), albeit with different efficiencies. Compared to the first set of BACTH assays (Fig. 1a), these data suggest that a free N terminus of YmgF is critical for its dimerization and its interactions with many Fts proteins. No complementation was detected with the polytopic membrane protein MalG, a component of the E. coli maltose transport system (20). In addition, we also tested interactions of YmgF with chimeric FtsL proteins, in which either the cytosolic domain (FLL) or the transmembrane segment (LFL) of FtsL were replaced with a cytosolic or transmembrane segment of MalF, another component of the E. coli maltose transporter (20, 33). YmgF was able to associate with the chimeric FtsL(FLL) but not with FtsL(LFL), suggesting that the FtsL transmembrane segment was crucial for YmgF interaction with FtsL (Fig. 1).

It should be noted that coexpression of the YmgF-T18 hybrid with the various T25-Fts proteins affected the growth of the bacterial cells compared to bacterial cells coexpressing the same T25-Fts fusions and the T18-YmgF hybrid: the plates had to be incubated for 3 or 4 days to obtain large enough colonies to measure β-galactosidase enzymatic activity. Furthermore, cells were differently affected depending upon the combination of the expressed hybrid proteins. The most drastic effect was observed upon coexpression of YmgF-T18 with T25-FtsQ, which turned out to be highly toxic for the cells. The origin of this toxicity is unclear, although it was not observed when YmgF-T18 was coexpressed with T25 fused to a truncated variant of the FtsQ lacking the last 30 C-terminal residues (data not shown). This truncated FtsQ1-246 was previously reported to be nonfunctional and unable to interact with and recruit "downstream" Fts partners to the septum (13, 37). On the basis of these data, we suggest that the toxicity of the T25-FtsQ/YmgF-T18 complex may be directly linked to the integrity and the functionality of the FtsQ moiety in this assembly. However, no drastic toxicity was observed when we coexpressed the YmgF-T25 and T18-FtsQ hybrid proteins.

In summary, the BACTH complementation results showed that YmgF, isolated as a partner of FtsL, could also efficiently associate with many other E. coli Fts proteins, thus suggesting that it might be part of the cell division apparatus.

YmgF is expressed in E. coli cells. The ymgF gene is located in a 2,270-bp "intergenic" region of the E. coli chromosome between a conserved gene (ycgG) and a pseudogene (ycgH1) (41). As a first step, we examined whether ymgF was expressed in the E. coli cells. Bacterial promoter recognition programs (E. coli promoter map from nostradamus.cs.rhul.ac.uk/vigen (30); BPROM from SoftBerry, Inc.) predicted a putative promoter within a 60-bp DNA fragment immediately upstream from the ymgF ORF start codon. To demonstrate a potential promoter activity within this ymgF DNA fragment, we used a lacZ transcriptional reporter system (44). To accomplish that, a 96-bp DNA fragment, encompassing the putative promoter region and the first two codons of ymgF ORF, was cloned upstream of a promoterless lacZ gene in the pTL61T vector (44). E. coli NM554 cells were transformed with the resulting plasmid, pYmgF-lac, and the transformants were plated on LB agar containing X-Gal. Quantitative analysis of promoter activity was done on overnight liquid cultures (see Materials and Methods). The level of β-galactosidase activity in NM554 cells carrying pYmgF-lac plasmid (24,000 U/mg [dry weight] bacteria) was eightfold higher than that measured in cells carrying the parental plasmid pTL61T (3,200 U/mg [dry weight]). This result demonstrates the presence of an active promoter region within the 90-bp fragment upstream of the ymgF ORF start codon. Reverse transcription-PCR experiments carried out on total mRNA from the E. coli MG1655 strain not only confirmed the expression of a ymgF mRNA under standard growth conditions but also indicated that ymgF does not form an operon with the downstream pseudogene ycgH1 (data not shown).

YmgF is a protein with two transmembrane segments. The membrane topology of YmgF was predicted in silico (5, 37) to contain two transmembrane segments separated by a short periplasmic loop and with both the N- and C-terminal extremities in the cytosol (Fig. 2a). To validate the theoretical model, we experimentally determined the membrane topology of YmgF using a dual pho-lac reporter system, which consists of the E. coli alkaline phosphatase fragment PhoA22-472 fused in frame with the {alpha}-peptide of E. coli β-galactosidase, LacZ4-60 (4). In this fusion approach, a periplasmic or membrane location of the reporter leads to high alkaline phosphatase activity and low β-galactosidase activity, whereas a cytosolic location of the reporter results in high β-galactosidase activity and low alkaline phosphatase activity. In-frame insertions of the pho-lac reporter after seven selected ymgF codons (D8, M21, K32, E39, N48, L57, and Q72) were constructed in plasmid pKTop (see Materials and Methods). E. coli DH5{alpha} was then transformed with the plasmids expressing the different YmgF'/Pho-Lac fusions, and the resulting transformants were analyzed on a dual-indicator LB medium containing both a blue chromogenic substrate for phosphatase activity (X-Phos) and a red chromogenic substrate for β-galactosidase activity (Red-Gal). As expected from the bioinformatics data, the cells expressing the YmgFD8-PhoLac or YmgFQ72-PhoLac fusions exhibited a red phenotype (i.e., Lac+), indicating a cytosolic location of the Pho-Lac reporter and, consequently, of the corresponding residues D8 and Q72 of YmgF (Fig. 2b). Insertion of the Pho-Lac reporter after residue L57 of YmgF yielded red-purple colonies on the dual-indicator medium, suggesting that residue L57 is localized in the cytosol rather than in the membrane, as predicted by three of the bioinformatics analyses (Fig. 2). This L57 residue, therefore, likely corresponds to the extremity of the second transmembrane domain of YmgF. When the Pho-Lac reporter was fused after residue M21, K32, E39, or N48 of YmgF, the hybrid proteins exhibited high phosphatase activity (deep blue phenotype), consistent with a periplasmic or transmembrane location of the corresponding YmgF residues (Fig. 2b).


Figure 2
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  		FIG. 2. YmgF topology analysis. (a) In silico models of the membrane topology of YmgF. Predictions 1, 2, and 3 were made using the software programs from the PONGO server; prediction 4 was made with software from the PSIPRED protein structure prediction server. YmgF residues are colored according to their localization relative to the inner membrane: red for cytosolic residues, light blue for transmembrane segments (transmembrane segment 1 [TMS1] and TMS2), and deep blue for periplasmic residues. The small black arrowheads on the top of the sequences indicate the positions of different fusions with the Pho-Lac reporter. (b) Experimental determination of YmgF membrane topology. E. coli DH5{alpha} cells expressing the different YmgF'-Pho-Lac fusions (position of insertion indicated in the label) were plated on indicator medium with two chromogenic substrates, Red-Gal (for β-galactosidase activity) and X-Pho (for phosphatase activity). Blue coloration of the colonies (high phosphatase activity) indicates a membrane or periplasmic location of the fusion point. Red coloration of the colonies (high β-galactosidase activity) indicates cytosolic location of the fusion point. Control cells (i.e., E. coli DH5{alpha}/pKTop) are indicated by the C label.

Altogether, the dual Pho-Lac experimental approach confirmed that YmgF is a polypeptide possessing two transmembrane segments, encompassing residues 10 to 30 and 39 to 57, separated by a short periplasmic loop (residues 30 to 38), and with both termini exposed to the cytosol. The membrane association of YmgF was further corroborated by subcellular fractionation of cells expressing a YmgF-GFP fusion of 37 kDa, which was found to be fully associated with the bacterial membrane fraction (Fig. 3). These results confirmed that YmgF is an integral membrane protein.


Figure 3
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  		FIG. 3. Membrane localization of YmgF-GFP. Analysis of protein from the soluble (S), insoluble (I), and membrane (M) fractions or from the total cell extract (TE) prepared from E. coli MG2007 containing a YmgF-GFP fusion or from E. coli MG1655, the parental strain (see Materials and Methods). The proteins were resolved by SDS-polyacrylamide gel electrophoresis (10%), electrotransferred to a polyvinylidene difluoride membrane, and probed with anti-GFP antibodies. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the gel, and the position of the YmgF-GFP protein is indicated by the arrow to the right of the gel.

YmgF localizes to the cell septum. To examine whether YmgF might be recruited to the cell division site under standard growth conditions, we constructed a recombinant strain, MG2006, that harbored an ymgF-gfp translational fusion, coding for a green fluorescent protein variant (GFP28) fused to the C terminus of YmgF. This gene fusion was integrated into the chromosome of MG1655 at the lambda attachment site attB (see Materials and Methods). Expression of the YmgF-GFP fusion was driven by an IPTG-inducible promoter. We also constructed MG2007, a derivative of the MG2006 merodiploid strain (ymgF+/ymgF-gfp), by deleting the wild-type copy of ymgF (replaced by a kanamycin-resistant cassette). Western blot analysis with anti-GFP antibodies indicated that the expressed YmgF-GFP fusion protein was stable and fully associated with the membrane fraction (Fig. 3).

Subcellular localization of the YmgF-GFP protein was examined by fluorescence microscopy analysis of strains MG2006 and MG2007, grown in minimal medium in the presence of 100 µM of IPTG until early exponential phase. Under these conditions, the expression of YmgF-GFP did not interfere with the division process, as the cells displayed wild-type morphology. A distinct fluorescent signal at midcell could be observed in approximately 30% to 40% of the bacteria. As shown in Fig. 4, the YmgF-GFP fusion was clearly localized at the septum in dividing cells. Importantly, the majority of the cells, in which the recombinant YmgF-GFP protein accumulated at the septal ring, had constrictions, whereas cells without a visible constriction rarely displayed a fluorescence band at midcell (data not shown). Noticeably also, the fluorescent signal at midcell tended to concentrate to a single dot in cells displaying a highly constricted septum. The YmgF-GFP septal localization disappeared before physical separation of the cells, and the fluorescent signal was found to be regularly spread along the membrane. No difference in the localization of YmgF-GFP between MG2006 and MG2007 strains was detected (Fig. 4).


Figure 4
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  		FIG. 4. Septal localization of YmgF-GFP. E. coli MG2006 cells (expressing both the wild-type YmgF protein and a YmgF-GFP fusion) (a) or MG2007 cells (expressing only the YmgF-GFP fusion but not the wild-type YmgF protein) (b) were grown at 30°C in minimal medium in the presence of 100 µM IPTG and examined by fluorescence microscopy. Representative GFP fluorescence images of exponentially growing cells are shown in the left panels. The phase-contrast images and the corresponding fluorescence images of the selected cells are shown in the middle and right panels, respectively. The arrows point to the septal localization of YmgF-GFP in the dividing cells. A statistical analysis of YmgF-GFP localization was performed on several independent cultures: out of a total of 217 scored cells, 69 (32%) exhibited a distinct septal localization, and among these cells, 49 (70%) had a visible constriction, indicating that they had initiated septation. The remaining cells showed a membrane-distributed fluorescence signal.

The YmgF localization pattern (i.e., a rather low percentage [30 to 40%] of exponentially growing bacteria with YmgF-GFP localized at the septum and the majority of these undergoing septation) appeared to be similar to that described for other E. coli cell division proteins, such as FtsN (2) or AmiC (9), suggesting that, like FtsN and AmiC, YmgF might be recruited to the cell division apparatus at a late stage of septum formation. For comparison, it has been reported that in the similar growth phase, 80 to 90% of the cells displayed midcell localization of the so-called early recruited proteins FtsZ (GFP-FtsZ) or ZapB (ZapB-GFP) (1, 19).

We also tested the septal localization of a GFP-YmgF hybrid protein, in which the GFP polypeptide was fused to the N terminus of YmgF. Although this protein exhibited a clear membrane distribution, it was only rarely localized to the septum (in about 3 to 4% of bacterial cells) (data not shown). These results, along with the BACTH interaction assays, suggest that YmgF may require a free N terminus to efficiently assemble into the septosome.

YmgF-GFP septal localization is dependent upon the presence of the FtsZ, FtsA, FtsQ, and FtsN cell division proteins. To check whether the recruitment of YmgF-GFP to the cell division site was dependent upon the presence of other Fts proteins, we analyzed the septal localization of the fusion in strains that could be selectively depleted in one of the following essential E. coli cell division proteins, FtsZ, FtsA, FtsQ, or FtsN.

Thermosensitive alleles of ftsZ (ftsZ84), ftsA (ftsA12), and ftsQ (ftsQ1) genes were introduced into MG2006 cells carrying the ymgF-gfp fusion by P1 transduction to yield strains MG06Zts, MG06Ats, and MG06Qts, respectively. Unexpectedly, the MG06Qts cells were found to exhibit a filamentous phenotype even at a permissive temperature (30°C) and turned out to be sensitive to low osmolarity (data not shown). Therefore, we constructed another derivative of MG2006, MG06Qnull (ftsQE14::kan), containing also a plasmid with a wild-type copy of ftsQ under the control of an arabinose-dependent PBAD promoter. Similarly, we constructed MG06Nnull (ftsN::kan), containing also a plasmid with a wild-type copy of ftsN under the control of the PBAD promoter. In both strains, MG06Qnull and MG06Nnull, the depletion of FtsQ or FtsN could be triggered by replacing arabinose by glucose in the growth medium.

As seen in Fig. 5, when these strains were grown in permissive conditions, that is, at 30°C for strains MG06Zts and MG06Ats or in the presence of arabinose for strains MG06Qnull and MG06Nnull, cells displayed a wild-type morphology (although MG06Qnull cells became slightly elongated). The YmgF-GFP fusion was found to be localized to the septum of dividing cells. In contrast, when the strains were grown under nonpermissive conditions, that is, at 42°C for strains MG06Zts and MG06Ats and in the presence of glucose for strains MG06Qnull and MG06Nnull, cells exhibited a filamentous phenotype, and a discrete fluorescent signal of the YmgF-GFP fusion could barely be detected (Fig. 5).


Figure 5
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  		FIG. 5. YmgF-GFP septal localization is dependent upon FtsZ, FtsA, FtsQ, and FtsN. The GFP fluorescence images of E. coli strains grown under permissive conditions are shown in the top images. The strains grown under nonpermissive condition (i.e., at 42°C for strains MG06Zts and MG06Ats or in the presence of glucose for strains MG06Qnull and MG06Nnull) are shown in the bottom images.

These results show that YmgF targeting to the cell septum is dependent upon the presence of FtsZ, FtsA, FtsQ, and FtsN and probably upon other E. coli cell division proteins, such as ZipA, FtsK, FtsL, FtsB, FtsI, FtsW, and FtsI (that are required for FtsN septal localization). Hence, YmgF appears as a late recruit to the septal ring. In addition, we found that the septal localization of YmgF was independent of the presence of the nonessential cell division components, AmiC, EnvC, TolA, TolQ, and Pal proteins, that are also recruited to the septum in the FtsN-dependent manner (see Fig. S1 in the supplemental material).

YmgF overcomes the thermosensitivity of an E. coli ftsQ1(Ts) mutant and restore its viability under low-osmolarity conditions. Prior data have shown that YmgF is not essential for cell viability under standard growth conditions (LB medium at 37°C). We confirmed that the growth and morphology of strain JW1156 (Keio collection), which harbors a full-length deletion of YmgF ({Delta}ymgF::kan), were comparable to those of the parental strain BW25113 in any of the growth conditions tested (temperature of 30, 37, or 42°C; rich or minimal medium; and low or high osmolarity) (data not shown). However, when the same deletion ({Delta}ymgF::kan) was P1 transduced into E. coli MG1665, the resulting strain (MG2005) exhibited a significant growth defect at 37°C or 42°C, but not at 30°C, compared to the parental strain MG1665. Yet, this growth defect was not rescued by expression of YmgF from a pUC19-ymgF plasmid. Other genetic loci within the ymgF region may be associated with the growth defect of the MG1665 transductants at high temperatures. The ymgF null mutation was also transduced into strain JOE309 (13). To rule out the possibility of acquisition, during strain construction, of an unlinked suppressor that would obscure a potential phenotype resulting from the ymgF deletion, the {Delta}ymgF::kan allele was P1 transduced into the JOE309 strain carrying a plasmid expressing YmgF under the control of an arabinose-dependent promoter (pBAD33-ymgF). No phenotypic difference could be evidenced between the parental strain and the resulting transductants depleted of YmgF (data not shown). Hence, from these studies, no noticeable phenotype could be attributed to the deletion of YmgF.

To further identify a putative role of YmgF in cell division, we explored the effect of YmgF overexpression on the phenotype of the following E. coli fts thermosensitive mutant strains: MCZ84 [ftsZ(Ts)], MCA12 [ftsA(Ts)], MCI23 [ftsA(Ts)], and MCQ1 [ftsQ(Ts)]. YmgF, when overproduced, could partially overcome the thermosensitivity of the ftsQ1(Ts) mutant (Fig. 6), but not that of ftsZ(Ts), ftsI(Ts), or ftsA(Ts) mutant (data not shown). YmgF could also overcome the severe growth defect of the ftsQ1(Ts) mutant when it was grown at a permissive temperature in low-osmolarity medium (Fig. 6). However, YmgF could not rescue the growth of strain JOE417 (ftsQE14::kan) fully depleted of FtsQ when cultured in the presence of glucose (data not shown).


Figure 6
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  		FIG. 6. Overproduced YmgF overcomes the thermosensitivity and growth defect of an ftsQ1(Ts) mutant. (a and c) Viability assays. E. coli MCQ1(pUC19) and MCQ1(pUC19-ymgF) cells were grown overnight in LB broth at 30°C. The cultures were diluted 1:200 in fresh LB and further incubated at 30°C for 3 to 4 h. (a) After adjustment of cell densities, 10-µl aliquots of serial dilutions were spotted on LB agar plates (plus ampicillin) that were incubated either at 30°C for 48 h (left) or at 42°C for 24 h and then at 30°C for 24 h (right). The serial dilutions 100, 10–1, 10–2, 10–3, 10–4, 10–5, and 10–6 are indicated by the 0, –1, –2, –3, –4, –5, and –6, respectively, at the sides of the panels. Alternatively (c), to check sensitivity to osmolarity, 10-µl aliquots of the same serial dilutions were spotted on LB0 or on LB1 agar (plus ampicillin) and incubated at 30°C for 24 h. (b and d) Microscopic observations. Overnight cultures of E. coli MCQ1(pUC19) and MCQ1(pUC19-ymgF) grown in LB at 30°C were diluted (1:200) into fresh LB and further incubated at 30°C for 3 to 4 h. Bacteria were then diluted 1:2 into prewarmed LB and incubated at 42°C for 2 h before analysis by phase-contrast microscopy (b). Alternatively (d), bacteria were observed after an overnight culture at 30°C in either LB0 or LB1 broth (plus ampicillin).

Altogether, these data suggest that by interacting with FtsQ, YmgF might participate in the stabilization of the cell divisome under specific conditions.


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DISCUSSION
 
We report here the characterization of YmgF, an inner membrane protein of 72 residues that specifically associates with the E. coli cell division machinery. YmgF was identified by BACTH screening as an interacting partner of the essential cell division protein FtsL. Systematic BACTH complementation assays revealed that, in addition to FtsL, YmgF was able to associate in vivo with many other Fts proteins, namely, FtsA, FtsB, FtsI, FtsQ, FtsN, FtsW, FtsX, and FtsZ. This suggested that YmgF might associate with the cell division apparatus. Indeed, analysis of the subcellular localization of YmgF with a fluorescently tagged molecule (YmgF-GFP) revealed that YmgF was localized to the septum in dividing cells. The septal targeting of YmgF was dependent upon the presence of several cell division proteins, including FtsZ, FtsA, FtsQ, and FtsN, indicating that YmgF can associate with the cell division apparatus only when this machinery is fully assembled.

Altogether, these results suggest that YmgF might be a novel component of the E. coli cell division machinery, although at this time, we have no clue about the potential physiological function of this component. Two observations may bring into question the physiological implication of this protein in the cell division process. First, YmgF appears to be present only in E. coli strains (except in enterohemorrhagic E. coli strains) and in Shigella flexneri. It is likely, however, that given its small size and the fact that this polypeptide is mainly constituted of two transmembrane segments, putative YmgF orthologs in other species might be sufficiently divergent to be unrecognized on the basis of sequence similarity. It is also noteworthy that many of the essential Fts proteins are poorly conserved (30, 41, 51, 52). Second, YmgF is a nonessential protein, as several studies showed that it could be inactivated without affecting cell viability (6, 23, 31). No obvious phenotype (growth rate, survival, morphology, etc.) associated with either deletion or overproduction of YmgF could be evidenced thus far. Yet, there are many proteins known to associate with the cell division machinery that are also not essential for cell viability or are required for cell growth only under specific conditions. For example, the cell division proteins MinC, ZapA, EzrA, and SulA that are known to directly interact with FtsZ and influence its function in vivo are not essential for cell survival (34). AmiC and EnvC, the septal murein hydrolases that facilitate the separation of daughter cells are also dispensable for viability (9, 10). Other components, such as FtsX, FtsE, and FtsP (SufI), become essential for the cell division process only when the bacteria are exposed to certain stress conditions (47, 48, 50). Furthermore, many essential division proteins are also dispensable for cell survival under particular physiological conditions. For example, overproduction of FtsN is sufficient to rescue thermosensitive alleles of several genes encoding essential proteins, such as FtsA (ftsA12), FtsQ (ftsQ1), FtsI (ftsI23) (15), and FtsK (ftsK44) (18), or even the complete deletions of ftsK (22, 29) or the ftsE and ftsX genes (47). Recently, it has been reported that gain-of-function mutations in FtsA could bypass the requirement of FtsN, ZipA, or FtsK (8, 21, 22). Interestingly, we found that YmgF, when overproduced, could partially overcome thermosensitivity and the severe growth defect at low osmolarity of an ftsQ(Ts) mutant. However, YmgF was unable to complement a total depletion of FtsQ. Other fts mutants, such as ftsZ(Ts), ftsA(Ts), and ftsI(Ts) mutants, were not affected by overproduction of YmgF. Taken together, these data suggest that YmgF could specifically stabilize the thermosensitive variant of FtsQ, likely through direct protein-protein interactions, thus allowing cell septation under nonpermissive conditions.

The fact that many of the so-called essential proteins can be dispensable for cell division under specific (albeit artificial) conditions (35) raises numerous questions regarding their biochemical functions. One emerging view is that these components might contribute specifically to the stabilization of the overall cell division machinery, although it is unclear how it is achieved at the molecular level. One could speculate that the YmgF polypeptide, identified here as an interacting partner of many Fts proteins and specifically recruited to the septum, could also contribute to the stabilization of the divisome. YmgF might become important for the assembly of a functional septosome only under particular physiological conditions or in the absence of certain cell division components, as illustrated here by the ability of YmgF to rescue growth of a thermosensitive ftsQ(Ts) mutant strain under nonpermissive conditions. Interestingly, YmgF was found to be recruited to the septum in an FtsN-dependent manner, suggesting that it could associate only with the fully assembled cell division apparatus. Furthermore, the fluorescent YmgF-GFP fusion was detected at midcell only in bacteria displaying a clear constriction. YmgF, therefore, might be a late recruit to the septal ring and could participate to the very terminal phase of the septation process leading to the closing of the septal ring under certain circumstances. It could play a role as a checkpoint to signal the completion of the divisome assembly before proceeding to the separation of the two daughter cells.

The promiscuous binding of YmgF with many Fts proteins is intriguing, especially when considering the small size of the polypeptide. One possible explanation is that, as the BACTH assay is carried out with hybrid proteins that are overproduced compared to the native expression levels of Fts proteins, this technique may reveal interactions of low affinity between YmgF and its Fts protein partners. Under native conditions (i.e., at lower level of expression), such associations may occur only in the fully assembled septosome. This could be the reason why all the components of the septosome are necessary for YmgF septal targeting. Another possibility (not exclusive) is that YmgF uses the same binding surfaces (or partly overlapping) to interact with different partners at different time points in the cell cycle. Yet, as discussed in our previous study (38), one cannot formally exclude the possibility that some of the detected complementation between YmgF and the E. coli divisome proteins may, in fact, result from indirect interactions.

Finally, it should be noted that the BACTH library screens reported here were certainly far from being exhaustive, especially as we did not isolate the proteins previously shown to efficiently interact with FtsL, such as FtsB, FtsQ, and FtsN (38). We are currently constructing libraries of higher complexity to further characterize the E. coli cell division interactome.


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ACKNOWLEDGMENTS
 
We thank Nienke Buddelmeijer, Jon Beckwith, Thomas Linn, and Mary Berlyn (E. coli Genetic Stock Center) and the National BioResource Project (NIG, Japan) for providing us with various E. coli strains and plasmids. We are greatly indebted to Agnes Ullmann for helpful discussions, insightful comments on the manuscript, and encouragement. We are grateful to Maryline Davi for technical assistance with plasmid constructions and other members of the Ladant lab for their interest.

This work was supported by the Institut Pasteur and Centre National de la Recherche Scientifique (CNRS URA 2185, Biologie Structurale et Agents Infectieux). C.R. holds a Marie Curie Outgoing International Fellowship (contract MOIF-CT-2005-008977).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Structural Biology and Chemistry, Institut Pasteur, 28 rue du Docteur Roux, Paris 75015, France. Phone: 33145688400. Fax: 33140613042. E-mail: ladant{at}pasteur.fr Back

{triangledown} Published ahead of print on 31 October 2008. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, January 2009, p. 333-346, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.00331-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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