Regulation and SOS induction of division inhibition in Escherichia coli K12

Summary When Escherichia coli is subjected to treatments that damage DNA or perturb DNA replication considerable cell filamentation occurs. It has been postulated that this phenomenon is associated with the presence of a division inhibitor induced coordinately with the SOS functions. The role of this induction would be to delay septation during DNA repair to prevent the formation of DNAless cells. In this communication, we present evidence for such a division inhibitor based on the properties of a division mutant which is hyperactive in the septation delay. Cells of this mutant filament extensively after a nutritional shift-up, have drastically reduced colony-forming abilities on a rich medium but not on a minimal medium following treatment with ultraviolet radiation and, are deficient in the lysogenization of phage lambda; phenotypes which are characteristic of but expressed to a much lower extent in another type of division mutant called lon. Cells harboring the division mutation plus either one of the lexA mutant alleles, spr-51 or tsl-1, are filamentous suggesting that they are permanently derepressed for division inhibition. These results are in agreement with models that assign the regulation of cell division to a division inhibitor which is regulated by the lexA repressor protein.     

Characterization of ftsZ, the Cell Division Gene of Buchnera aphidicola (Endosymbiont of Aphids) and Detection of the Product

Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division. With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont. Nucleotide sequence determination of a 6.4-kilobase B. aphidicola DNA fragment has indicated that, as in E. coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC–ddlB–ftsA–ftsZ). Although B. aphidicola ftsZ is expressed in E. coli, it cannot complement E. coli ftsZ mutants. High levels of B. aphidicola FtsZ results in the formation of long filamentous E. coli cells, suggesting that this protein interferes with cell division. The presence of FtsZ indicates that in this, as well as in many other previously described properties, B. aphidicola resembles free-living bacteria. Received: 22 July 1997 / Accepted: 28 July 1997     

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto‐ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto‐ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZ_L169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZ_L169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain‐of‐function mutants such as FtsA* or increased levels of the FtsA‐interacting protein FtsN. Similar to FtsA*, FtsZ_L169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZ_L169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Induction of only limited elongation instead of filamentation by inhibition of cell division in Corynebacterium glutamicum

In order to characterize the cell-division mechanism of coryneform bacteria, we tried to isolate cell-division mutants from Corynebacterium glutamicum after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, such as Escherichia coli fts mutants, which form long filaments at the restrictive temperatures. At the non-permissive temperature, most of the mutants formed club-shaped or dumbbell-shaped, elongated rod cells, but no filament formers were isolated. Then we examined the effects of cell division inhibitors on this organism. Cephalexin and sparfloxacin, which are the inhibitors of septation and DNA synthesis respectively, and are known to cause cell filamentation in E. coli, did not cause filamentation in C. glutamicum but induced morphological changes that were similar to those observed with the temperature-sensitive ts mutants of C.␣glutamicum. These results suggest that C. glutamicum has a unique regulation mechanism, that is, the inhibition of cell division leads to cessation of cell elongation. Received: 5 February 1998 / Received revision: 6 April 1998 / Accepted: 27 April 1998     

Cell division in a minimal bacterium in the absence of ftsZ

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non‐essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.     

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis

In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its β-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher β-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.     

Effect of Mechanical Vibrations on the lonMutant of Escherichia coliK-12

It was found that, depending on their frequency, mechanical vibrations (MVs) can either stimulate (4 Hz) or inhibit (50 Hz) the growth and the division of the lonmutant of Escherichia coliK-12. Similar effects were observed when the MV-treated nutrient medium was inoculated with untreated mutant cells. MVs enhanced the motility of mutant cells and the fragmentation of filament cells always present in the populations of lonmutants.     

The sfiA11 mutation prevents filamentation in a response to cell wall damage only in a recA+ genetic background

Summary N--palmitoyl-l-lysyl-l-lysine dihydrochloride ethyl ester (PLL) at sublethal doses causes filamentous growth of E. coli strains except sfiA mutants, which divide normally in its presence. PLL does not elicit the SOS responses as judged by prophage induction, an increase of RecA protein synthesis or induction of the sfiA operon in a sfiA::lacZ fusion strain. Thus, it appears that filamentation caused by PLL is not an SOS function and might be the result of membrane damage by PLL, which is an amphipathic compound and at higher doses causes cell lysis. This indicates that basal levels of the sfiA gene product are sufficient to inhibit cell division in the presence of PLL.We have found further that the phenotype of the sfiA mutation in the presence of PLL requires a recA ⁺ genetic background and does not occur in E. coli recA1 sfiA11, recA13 sfiA11, recA56 sfiA11 and recA441 sfiA11. All these strains, but rec441 sfiA11, however, regain the ability of sfiA11 mutants to divide in the presence of PLL after transformation with the RecA overproducing-plasmid pXO₂. This supports the conclusion that the RecA protein positively affects sfiA11-mediated cell division in the presence of the cell membrane damaging compound, PLL. The basal level of the RecA protein in the recA ⁺sfiA11 strain is sufficient for this process. An increased level due to overproduction from the multicopy plasmid pXO₂ exerts the same effect.     

FtsI and FtsW Are Localized to the Septum in Escherichia coli

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.     

Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation

An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ⁺ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.     

A new mutation rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants

Summary We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon transfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45°C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9° C lower than does the wild type.The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E. coli RNA polymerase.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50?kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors.     

Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.     

Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ

A homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor. The gene was located on the physical map of the chromosome at about ‘11 o'clock’ (in the vicinity of glkA, hisA and trpB). Surprisingly, a null mutant in which the 399-codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation. This indicates that cell division may not be essential for the growth and viability of S. coelicolor. The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores.