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.     

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.     

Role of sulA and sulB in filamentation by lon mutants of Escherichia coli K-12.

Cells containing the pleiotropic Escherichia coli mutation lon filament extensively and die after exposure to ultraviolet light. Outside suppressors of the ultraviolet sensitivity, called sul, have previously been described at two loci; these mutations reverse the ultraviolet sensitivity of lon strains but do not affect the mucoidal or degradation defect of these strains. An isogenic set of strains carrying combinations of lon, sulA, and sulI was constructed, and their behavior during normal growth and after ultraviolet treatment was studied. sulA mutations had no detectable phenotype in lon+ cells; the lon sulA strains filamented transiently after ultraviolet irradiation, as did lon+ sul+ cells. We found that the sulB mutation, which alters cell morphology and slows recovery from transient filamentation after ultraviolet treatment, was epistatic to both lon and sulA. Whereas sulA mutations were recessive to the wild-type allele, sulB was partially dominant. The simplest model to account for our observations is that sulA and lon participate in a pathway of filamentation independent of that which produces transient filamentation in wild-type strains; sulB product may be the target of sulA action and may play a role in normal cell division.     

Interaction between the min locus and ftsZ.

In Escherichia coli, distinct but similar minicell phenotypes resulting from mutation at the minB locus and increased expression of ftsZ suggested a possible interaction between these genes. A four- to fivefold increase in FtsZ resulting from increased gene dosage was found to suppress the lethality of minCD expressed from the lac promoter. Since increased MinCD did not affect the level of FtsZ, this suggested that MinCD may antagonize FtsZ to inhibit its cell division activity. This possibility was supported by the finding that alleles of ftsZ isolated as resistant to the cell division inhibitor SulA were also resistant to MinCD. Among the ftsZ(Rsa) alleles, two appeared to be completely resistant to MinCD as demonstrated by the lack of an effect of MinCD on cell length and a minicell phenotype observed in the absence of a significant increase in FtsZ. It was shown that SulA inhibits cell division independently of MinCD.     

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.     

Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.     

The ATP-Dependent HslVU/ClpQY Protease Participates in Turnover of Cell Division Inhibitor SulA in Escherichia coli

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [DeltahslVU and Delta(clpPX-lon)], cannot grow at low (30 degrees C) or very high (45 degrees C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30 degrees C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Delta(clpPX-lon) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

Isolation and characterization of ftsZ alleles that affect septal morphology.

The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery.     

Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.     

Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli.

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.     

Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.     

FtsZ dimerization in vivo

A hybrid assay, based on the properties of the lambda repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the lambda cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional lambda repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ.     

Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex.

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.     

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 .     

Protein engineering with synthetic Escherichia coli amber suppressor genes

We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.