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.     

Coupling of DNA replication and cell division: sulB is an allele of ftsZ.

Treatments that damage DNA in Escherichia coli result in the inhibition of cell division. This inhibition is controlled by the lexA-recA regulatory circuit and can be specifically uncoupled by the mutations sulA (sfiA) and sulB (sfiB), which map at 21 and 2 min, respectively. Presently it is thought that sulA codes for an inducible inhibitor of cell division, the expression of which is controlled directly by the lexA repressor. In this report, it is shown that sulB is an allele of ftsZ, an essential cell division gene. A sulB mutation leads to an altered ftsZ gene product which is slightly thermosensitive and has an altered mobility on polyacrylamide gels. It is suggested that the altered ftsZ gene product is resistant to the sulA inhibitor, thus permitting cell division after induction of the SOS response. It is also shown that an increase in the gene dosage of ftsZ delays the onset of filamentation after SOS induction.     

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.     

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.     

A new essential gene of the 'minimal genome' affecting cell division

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.     

Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA).

In Escherichia coli, the ftsZ gene is thought to be an essential cell division gene. Several dominant mutations that make lon mutant cells refractory to the cell division inhibitor SulA, sulB9, sulB25, and sfiB114, have been mapped to the ftsZ gene. DNA sequence analysis of these mutations and the sfiB103 mutation confirmed that all of these mutations mapped within the ftsZ gene and revealed that the two sulB mutations were identical and by selection for resistance to higher levels of SulA, contained a second mutation within the ftsZ gene. We therefore propose that these mutations be redesignated ftsZ(Rsa) for resistance to SulA. A procedure involving mutagenesis of ftsZ cloned on low-copy-number vectors was used to isolate three additional ftsZ(Rsa) mutations. DNA sequence analysis of these mutations revealed that they were distinct from the previously isolated mutations. One of these mutations, ftsZ3(Rsa), led to an altered FtsZ protein that could no longer support cell growth but still conferred the Rsa phenotype in the presence of ftsZ+. In addition to being resistant to SulA, all ftsZ(Rsa) mutations also conferred resistance to a LacZ-FtsZ hybrid protein (ZZ). One possibility is that FtsZ functions as a multimer and that FtsZ(Rsa) mutant proteins have an increased ability for multimerization, making them resistant to SulA and ZZ.     

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.     

ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

Regulation by hetC of genes required for heterocyst differentiation and cell division in Anabaena sp. strain PCC 7120

Unlike those of the wild-type strain, proheterocysts of the Anabaena sp. strain PCC 7120 hetC strain keep dividing. ftsZ, the most critical cell division gene, is up-regulated in hetC proheterocysts. Heterocyst differentiation genes hglD, hglE, patB, nifB, and xisA are no longer expressed in the hetC mutant. hetC also regulates the expression of patA, a pattern formation gene.     

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.