The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.

Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants. Among these, the ftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.     

Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts).

A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation. Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed. The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division. The ftsN gene was located at 88.5 min on the E. coli genetic map just downstream of the cytR gene. ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death. DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da. The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins. The presumed extracellular domain was unusual in that it was rich in glutamine residues. A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

FtsZ ring formation in fts mutants.

The formation of FtsZ rings (Z rings) in various fts mutants was examined by immunoelectron microscopy and immunofluorescence. In two temperature-sensitive ftsZ mutants which form filaments with smooth morphology, the Z ring was unable to form. In ftsA, ftsI, and ftsQ mutants, which form filaments with an indented morphology, Z rings formed but their contraction was blocked. These results indicate that fully functional ftsA, ftsQ, and ftsI genes are not required for Z-ring formation and are unlikely to have a role in localization of the Z ring. The results also suggest that one function of the Z ring is to localize the activity of other fts gene products.     

Cell shape and division in Escherichia coli: experiments with shape and division mutants.

Double mutants which carry mutations in genes (rodA, pbpA) required for cell elongation (i.e., maintenance of rod shape) in combination with mutations in genes (ftsA, ftsI, ftsQ, or ftsZ) required for septation were constructed. Such mutants were able to grow for about two mass doublings at a normal rate at the restrictive temperature (42 degrees C). The morphology of the cells formed under these conditions was interpreted by assuming the existence of a generalized system for peptidoglycan growth together with two additional systems which modify the shape of the growing peptidoglycan layer. The results also showed that different fts genes probably control different stages in septation. ftsZ (sulB or sfiB) appears to be required for the earliest step in septation, ftsQ and ftsI (pbpB or sep) are required for a later step or steps, and ftsA is required only for the latest stages in septation.     

MinCD-independent inhibition of cell division by a protein that fuses MalE to the topological specificity factor MinE.

We report that MinE, the topological specificity factor of cell division in Escherichia coli, inhibits septation when fused to the C terminus of the maltose-binding protein MalE. This contrasts with overexpression of MinE alone, which affects growth but has no effect on division. Inhibition by MalE-MinE was minCD independent and depended on MinE segments involved in dimerization and prevention of MinCD division inhibition. The SOS and the heat shock responses were not involved, suggesting that the inhibition comes from a direct interaction of MalE-MinE with the septation apparatus. MalE-MinE lethality was suppressed by overexpression of ftsZ, as well as by overexpression of ftsN, a suppressor of temperature-sensitive mutations in genes ftsQ, ftsA, and ftsI. We also report that high-level synthesis of MalE disturbs nucleoid partitioning.     

Regulation of cell division in Escherichia coli K-12: probable interactions among proteins FtsQ, FtsA, and FtsZ.

In Escherichia coli, the FtsQ, FtsA, and FtsZ proteins are believed to play essential roles in the regulation of cell division. Of the three proteins, FtsZ has received the most attention, particularly because of its interactions with SfiA. Double mutants which carry mutations located in the ftsQ, ftsA, or ftsZ gene in combination with the lon-1 mutation were constructed. In the presence of the lon-1 mutation, which is known to stabilize SfiA, the ftsQ1 mutant cells were not capable of forming colonies on a rich agar medium, whereas mutant cells harboring either one of the mutations grew well on this medium. Examination of lon-1 fts double-mutant cells for sensitivity to UV light revealed that those carrying the ftsA10 allele were resistant. It was also observed that in the presence of a multicopy plasmid containing a wild-type ftsZ gene, the ftsQ1 mutant filamented markedly following a nutritional shift-up and that the division rate of ftsZ84 mutant cells was slightly reduced when they harbored a wild-type ftsQ-containing plasmid. The possibility that the Fts proteins are interacting with one another and forming a molecular complex is discussed.     

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.     

Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB

Genes rcsC and rcsB form a two-component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB-dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co-regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.     

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes

We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.     

Borrelia burgdorferi ftsZ plays a role in cell division

ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZ(Bbu)). Its gene product (FtsZ(Bbu)) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZ(Bbu) might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZ(Bbu) antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZ(Bbu) could interfere with the function of E. coli FtsZ, ftsZ(Bbu) was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZ(Bbu) generated a filamentous phenotype. This suggested interference of ftsZ(Bbu) with E. coli FtsZ function and confirmed the role of ftsZ(Bbu) in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.