Characterization of YmgF, a 72-residue inner membrane protein that associates with the Escherichia coli cell division machinery

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.     

A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

Nucleoid-independent identification of cell division sites in Escherichia coli

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli

Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.     

Changes in cell diameter during the division cycle of Escherichia coli

Extensive measurements of steady-state populations of several Escherichia coli strains have consistently indicated that cell diameter decreases with increasing cell length. This was observed both after electron microscopy of air-dried cells and after phase-contrast microscopy of living cells. The analysis was made by considering separately the unconstricted cells and three classes (slight, medium, and deep) of constricted cells in the population. During slow growth, cells with the average newborn length were up to 8% thicker than unconstricted cells twice as long. This decrease in diameter is less at higher growth rates. Despite the small changes and the large variation of the diameter in any particular length class, significant negative correlations between diameter and length were obtained. Cell diameter increases again at the end of the cell cycle as indicated by an increase of average diameter in the three consecutive classes of constriction.     

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.     

The order of the ring: assembly of Escherichia coli cell division components

Topological cues appear to override temporal events in the assembly of the Escherichia coli cell division ring. When a procedure that allows the recruitment of ring components based on their topological properties is used, a concerted mode of assembly of several components of the divisome, rather than a strict linear mode, is revealed. Three multimolecular complexes, the proto-ring, the periplasmic connector and the peptidoglycan factory, show some degree of concertation for their assembly. In addition, back-recruitment of all late proteins except FtsN into the division ring occurs even in the absence of proteins incorporated at earlier stages, i.e. FtsA or FtsQ.     

FtsZ regulates frequency of cell division in Escherichia coli.

Cell division is regulated so that it occurs only once per cell cycle. In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole. Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length. We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place. This result suggests that the level of FtsZ controls the frequency of cell division in E. coli.     

Participation of the chemotactic system in regulating cell division in Escherichia coli

The possible role of the chemotaxis system in regulating cell division of Escherichia coli was studied. Attractants increased the rate of division whereas repellents reduced it. Non-metabolisable attractants analogues were also effective in stimulating cell division. Fucose, a non-metabolisable analogue of galactose, increased the rate of division by 20-25%. Co2+ at concentrations which had no effect on the tar-mutant division suppressed the division of the wild type. Likewise, indole at concentrations which did not influence the division of the tsr-mutant, suppressed the division of the wild type.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

Induction and stabilization of synchrony in the cell division of Escherichia coli

Escherichia coli strains B5 and B/r/1 were grown under conditions of periodic glucose starvation in a minimal medium. Such conditions of growth give rise to two synchronous populations that are out of phase regarding their time of division, one dividing shortly after a new supply of fresh medium and the other at a later stage of the feeding cycle. Preferential selection of one of the two populations using heat treatment resulted in a homogeneous synchronized culture that exhibited in a non-limiting medium a high degree of synchrony that was long lasting. Synchrony and its persistence could survive preservation of such a synchronized culture by freeze drying. An explanation of the synchrony persistence was put forward and the practical implications of these findings were discussed.     

Functional analysis of the cell division protein FtsW of Escherichia coli

Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in peptidoglycan assembly during cell elongation, division, and sporulation. This essential cell division protein has 10 transmembrane segments (TMSs). It is a late recruit to the division site and is required for subsequent recruitment of penicillin-binding protein 3 (PBP3) catalyzing peptide cross-linking. The results allow identification of several domains of the protein with distinct functions. The localization of PBP3 to the septum was found to be dependent on the periplasmic loop located between TMSs 9 and 10. The E240-A249 amphiphilic peptide in the periplasmic loop between TMSs 7 and 8 appears to be a key element in the functioning of FtsW in the septal peptidoglycan assembly machineries. The intracellular loop (containing the R166-F178 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information.     

Dynamic localization cycle of the cell division regulator MinE in Escherichia coli

The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites. MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion. MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves. Using fixed cells, we previously found that a MinE-green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring. Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation. The results show that MinE is not a fixed marker for septal ring assembly. Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave-like fashion. These waves run from a midcell position towards the poles in an alternating sequence such that the time-averaged concentration of division inhibitor is lowest at midcell.     

The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ

ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.     

Negative regulatory role of the Escherichia coli hfq gene in cell division

We found that the hfq::cat mutant strain produced minicells at high frequency. Minicell production by the mutant strain was more prominent in poor media and in the stationary phase than in rich media and in the exponentially growing phase. The amount of the cell division protein FtsZ increased up to two- to threefold of the wild-type cells in the hfq::cat mutant in the stationary phase, while such differences were not observed in the exponentially growing phase. Increased ftsZ mRNA levels were also observed in the hfq::cat mutant in the stationary phase. These results suggest a negative regulatory role of the DNA-, RNA-binding protein Hfq in cell division in the stationary phase.     

Role of the sfiA-dependent cell division regulation system in Escherichia coli.

Several authors have suggested that the SOS-associated (sfiA-dependent) system of division inhibition, normally induced by perturbations of DNA replication, also regulates steady-state (unperturbed) cell division. The present work shows that mean cell mass is identical in sfiA+ and sfiA mutant cultures during steady-state growth, that mass adjustment is identical after shift up, that sfiA expression is not induced by shift up, and that a sfiA mutation does not cause aberrant chromosome segregation.