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.     

Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli.

A dominant sfiB allele has been cloned which renders partial diploids of an sfiB + Escherichia coli host resistant to division inhibition mediated by the SOS response. Transpositional mutagenesis was used to map the position of this sfiB114 allele, carried by a plasmid pLG552 , to an approximately 0.6-kb region overlapping the coding regions for ftsA and ftsZ , two genes essential for normal division. Most Tn 1000 insertions which inactivated sfiB114 also inactivated the ftsA function and caused the disappearance of both a 47-K polypeptide and reduced levels of a 42-K polypeptide in maxi-cells carrying pLG552 . An additional insertion inactivating sfiB114 was mapped to the right of ftsA and resulted in loss of the 42-K but not the 47-K polypeptide in maxi-cells. Moreover, a 2.1-kb BamHI-EcoRI DNA fragment was subcloned which carried ftsA and coded for a 47-K polypeptide but did not carry sfiB114 and did not complement ftsZ . We conclude that sfiB114 is located within ftsZ coding for a 42-K polypeptide. Nevertheless, insertions into ftsZ coding the 47-K polypeptide suppress the sfiB114 allele by substantially reducing the synthesis of the FtsZ ( SfiB114 ) polypeptide. The level of residual FtsZ synthesis was minimal when Tn 1000 was inserted closest to the distal end of ftsA , indicating the presence of a regulatory region essential for maximal expression of ftsZ .     

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 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.     

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.     

Overlapping functional units in a cell division gene cluster in Escherichia coli

The ftsZ ( sulB ) coding sequence is preceded by two promoters, at least one of which lies within the coding sequence of the neighboring gene, ftsA . This region of the ftsA gene is required for full biological activity of ftsZ .     

Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity.

Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation. Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B. subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ. Previously, we cloned and sequenced the B. subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species. We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds. Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes. It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated. The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation. Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C. Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant. The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues).     

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.     

Enhanced production of recombinant proteins in Escherichia coli by filamentation suppression

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

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.     

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.     

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.