Inhibition of growth of ftsQ, ftsA, and ftsZ mutant cells of Escherichia coli by amplification of a chromosomal region encompassing closely aligned cell division and cell growth genes.

Amplification of a 2.6-kilobase chromosomal fragment of the mra region of Escherichia coli encompassing the ftsI(pbpB) gene and an open reading frame upstream with lethal to E. coli strains with mutations of the flanking cell division genes ftsQ, ftsA, and ftsZ. A shortened fragment in which the major portion of ftsI was deleted also had lethal effects on ftsQ and ftsZ mutants.     

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.     

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.     

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.     

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.     

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.     

Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli.

In Escherichia coli, the products of several genes are required for septation, and the products of several others are required for the maintenance of the rod shape of the cells. We show here that the combination of certain mutations in a division gene (ftsI) with a specific mutation in one of the shape genes (rodA) could produce cells with normal shape and division, although separately these mutations led to a loss of the capacity to divide (ftsI) or to form normal rod-shaped cells (rodA). In contrast, combinations between other mutant alleles of these genes produced double mutants which had lost the capacity both to divide and to form rod-shaped cells. The mutual phenotypic correction observed within particular pairs of mutant genes suggests that the normal morphogenetic cycle of growth and division may require direct interaction between the two membrane proteins which are the products of these genes.     

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell division gene ftsQ.

We report the identification, cloning, and mapping of a new cell division gene, ftsQ. This gene formed part of a cluster of three division genes (in the order ftsQ ftsA ftsZ) which itself formed part of a larger cluster of at least 10 genes, all of which were involved in some step in cell division, cell envelope synthesis, or both. The ftsQAZ group was transcribed from at least two independent promoters.     

Differential effect of mutational impairment of penicillin-binding proteins 1A and 1B on Escherichia coli strains harboring thermosensitive mutations in the cell division genes ftsA, ftsQ, ftsZ, and pbpB.

To study the functional differences between penicillin-binding proteins (PBPs) 1A and 1B, as well as their recently postulated involvement in the septation process (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989), a series of isogenic strains with mutations in the genes coding for PBP 1A (ponA) or PBP 1B (ponB) or in the cell division-specific genes ftsA, ftsQ, pbpB, and ftsZ was constructed and used as the start point to produce double mutants combining the ponA or ponB characters with mutations in cell division genes. PBP 1A seemed to be unable to preserve cell integrity by itself, requiring the additional activities of PBP 2, PBP 3, and FtsQ. PBP 1B was apparently endowed with a more versatile biosynthetic potential that permitted a substantial enlargement of PBP 1A-deficient cells when PBP 2 or 3 was inhibited or when FtsQ was inactive. beta-Lactams binding to PBP 2 (mecillinam) or 3 (furazlocillin) caused rapid lysis in a ponB background. The lytic effect of furazlocillin to ponB cell division double mutants was suppressed at the restrictive temperature irrespective of the identity of the mutated cell division gene. These results indicate that PBPs 1A and 1B play distinct roles in cell wall synthesis and support the idea of a relevant involvement of PBP 1B in peptidoglycan synthesis at the time of septation.     

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.     

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.     

Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci.

Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.     

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.     

Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants

Certain beta-lactam antibiotics induce the chromosomal ampC beta-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that beta-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an ftsZ mutant and other cell division mutants (ftsA, ftsQ, and ftsI) and a PBP2 mutant for induction of beta-lactamase. In all mutants, beta-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress beta-lactamase expression in vitro. Our results suggest that beta-lactamase is not induced because the fts mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.     

Impaired cell division and sporulation of a Bacillus subtilis strain with the ftsA gene deleted.

The ftsZ and ftsA genes of Bacillus subtilis are organized in a simple operon expressed from promoter sequences immediately upstream of ftsA. The promoter-distal ftsZ gene is an essential septation gene. In this report, it is shown that the promoter-proximal ftsA gene can be deleted in a previously constructed strain in which the essential gene, ftsZ, is under the control of the inducible spac promoter. Absence of the ftsA gene product resulted in a very filamentous morphology indicating an important role for ftsA in cell division. Also, growth was severely impaired, and viability and sporulation were reduced. The defective sporulation phenotype correlated with a deficiency in the processing of pro-sigma E to its active form.