Cell Division Gene ftsZ Research Articles

Light- and cytokinin-regulated ftsZ gene expression in excised cucumber cotyledons (Cucumis sativus)

Significant similarities are now known to exist between bacterial celldivision and the division of plastids in higher plants. This is primarily duetothe recently proven requirement of homologues of the bacterial cell divisiongene ftsZ, for plastid division. This report describes thegeneration of three partial FtsZ cDNAs from cucumber byreverse-transcriptase mediated- polymerase chain reaction (RT-PCR) and thesubsequent monitoring of the transcript levels of one ftsZgene by high sensitivity quantitative RT-PCR, in etiolated, excised cucumbercotyledon tissue in response to the exposure to light and the exogenousapplication of the phytohormones, cytokinin and auxin. The results obtainedclearly showed an increase in the transcript levels of theftsZ gene examined in response to exposure to light andtreatment with cytokinin, unlike in the case of auxin treatment, where nosignificant change was observed. These results are further discussed in termsofthe possible involvement of the ftsZ gene in light- andcytokinin- induced plastid division.

Construction of a xylanase-producing strain of Brevibacterium lactofermentum by stable integration of an engineered xysA gene from Streptomyces halstedii JM8.

A xylanolytic strain of Brevibacterium lactofermentum containing the Streptomyces halstedii His-tagged xysA gene was generated. The new strain contains DNA derived from S. halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division gene ftsZ. The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose. The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene. This strategy can be used to integrate any gene into the B. lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.

Expression of Neisseria gonorrhoeae cell division genes ftsZ, ftsE and minD is influenced by environmental conditions

The activity of the promoter regions of the cell division genes ftsZ, ftsE, minC, minD and minE from Neisseria gonorrhoeae (Ng) was studied under different environmental conditions using lacZ translational fusions. The promoters of the min Ng genes have not been previously determined and we identified promoter regions upstream of each gene ( minCp, minDp and minEp). We determined that minDp had the strongest activity. Expression of the promoter regions of ftsZ Ng and ftsE Ng, which we had previously identified, as well as minD Ng, were then studied under conditions reflecting the environment of the genitourinary tract. These conditions included anaerobiosis, presence of isoleucine or urea (3 mM and 400 mM, respectively) and acidity of pH 6. Both β-galactosidase expression and northern blot analysis indicated that all three genes were upregulated under anaerobiosis. The addition of isoleucine as well as media at pH 6 did not have any significant effects on the promoter activity of these genes while the presence of urea significantly decreased ftsZ Ng promoter activity. The expression of the minD Ng promoter region was analyzed during different growth phases and shown to follow the growth behavior of the culture. By contrast, the ftsZ Ng promoter activity continued to rise after the onset of the stationary phase. When gonococcal ftsZ promoter 1 (Pz1) was altered by site-directed mutagenesis, a significant decrease in the expression of ftsZ Ng was observed under both aerobic and anaerobic conditions. These data infer that gonococci regulate their cell division in response to different environments.

The cell division genes ftsQ and ftsZ, but not the three downstream open reading frames YFIH, ORF5 and ORF6, are essential for growth and viability in Brevibacterium lactofermentum ATCC 13869.

The three ORFs (YFIH, ORF5 and ORF6) located downstream of the cell division genes ftsQ and ftsZ in Brevibacterium lactofermentum were disrupted by single homologous recombination events between internal fragments of the corresponding genes and the chromosomal sequences. The phenotypes of the disrupted mutants were similar to that of the wild type, suggesting that these genes are dispensable for growth and viability. However, using different plasmid constructs, it was not possible to obtain disrupted ftsZ or ftsQ mutants by single crossover events. When the ftsZ or ftsQ gene sequence was disrupted in vitro and used to replace the homologous chromosomal gene by double recombination, only single recombination events took place, and therefore no disruptants were obtained. It may be concluded therefore that, as in Escherichia coli, the cell division genes ftsQ and ftsZ are indispensable for growth and viability of B. lactofermentum. Northern hybridisation analyses performed using internal fragments of the genes coding for YFIH, ORF5 and ORF6 allowed us to dissect their transcriptional organization and to confirm the disruption of these genes.

CtrA mediates a DNA replication checkpoint that prevents cell division in Caulobacter crescentus.

Coordination of DNA replication and cell division is essential in order to ensure that progeny cells inherit a full copy of the genome. Caulobacter crescentus divides asymmetrically to produce a non-replicating swarmer cell and a replicating stalked cell. The global response regulator CtrA coordinates DNA replication and cell division by repressing replication initiation and transcription of the early cell division gene ftsZ in swarmer cells. We show that CtrA also mediates a DNA replication checkpoint of cell division by regulating the late cell division genes ftsQ and ftsA. CtrA activates transcription of the P(QA) promoter that co-transcribes ftsQA, thus regulating the ordered expression of early and late cell division proteins. Cells inhibited for DNA replication are unable to complete cell division. We show that CtrA is not synthesized in pre-divisional cells in which replication has been inhibited, preventing the transcription of P(QA) and cell division. Replication inhibition prevents the activation of the ctrA P2 promoter, which normally depends on CtrA phosphorylation. This suggests the possibility that CtrA phosphorylation may be affected by replication inhibition.

Compartmentalization of transcription and translation in Bacillus subtilis.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.

We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

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.

Molecular evidence for single Wolbachia infections among geographic strains of the flour beetle Tribolium confusum.

Infections with the rickettsial microorganism Wolbachia are cytoplasmically inherited and occur in a wide range of insect species and several other arthropods. Wolbachia infection often results in unidirectional cytoplasmic incompatibility (CI): crosses between infected males and uninfected females are incompatible and show a reduction of progeny or complete inviability. Unidirectional CI can also occur when males harbouring two incompatible Wolbachia strains are crossed with females infected with only one of the two strains. In the flour beetle Tribolium confusum, Wolbachia infections are of particular interest because of the severity of incompatibility. Typically, no progeny results from the incompatible cross, whereas only partial incompatibility is observed in most other hosts. Werren et al. (1995a) reported that Wolbachia infections in T. confusum consist of two bacterial strains belonging to distinct phylogenic groups, based on PCR amplification and sequence analysis of the bacterial cell division gene ftsZ. However, Fialho & Stevens (1996) showed that eight strains of T. confusum were infected with a single and common incompatibility type. Here we report analysis of the ftsZ gene by specific PCR amplification. Diagnostic restriction enzyme assays revealed no evidence of double infections in 11 geographic strains of T. confusum, including the strain examined by Werren et al. (1995a). Further, sequence analysis of the Wolbachia ftsZ gene and an internal transcribed spacer (ITS) region in two of these strains displayed no nucleotide variation or evidence of polymorphisms. Results suggest that T. confusum is infected with B-group Wolbachia only.

Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli.

The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQ, and ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (approximately 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.

Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin

We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.

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.

Rhizobium meliloti contains a novel second homolog of the cell division gene ftsZ

We have identified a second homolog of the cell division gene, ftsZ, in the endosymbiont Rhizobium meliloti. The ftsZ2 gene was cloned by screening a genomic lambda library with a probe derived from PCR amplification of a highly conserved domain. It encodes a 36-kDa protein which shares a high level of sequence similarity with the FtsZ proteins of Escherichia coli and Bacillus subtilis and FtsZ1 (Z1) of R. meliloti but lacks the carboxy-terminal region conserved in other FtsZ proteins. The identity of the ftsZ2 gene product was confirmed both by in vitro transcription-translation in an R. meliloti S-30 extract and by overproduction in R. meliloti cells. As with Z1, the overproduction of FtsZ2 in E. coli inhibited cell division and induced filamentation, although to a lesser extent than with Z1. However, the expression of ftsZ2 in E. coli under certain conditions caused some cells to coil dramatically, a phenotype not observed during Z1 overproduction. Although several Tn3-GUS (glucuronidase) insertions in a plasmid-borne ftsZ2 gene failed to cross into the chromosome, one interruption in the chromosomal ftsZ2 gene was isolated, suggesting that ftsZ2 is nonessential for viability. The two ftsZ genes were genetically mapped to the R. meliloti main chromosome, approximately 100 kb apart.

Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein.

Escherichia coli divides by forming a septum across the middle of the cell. The biochemical mechanism underlying this process is unknown. Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E. coli cell division, ftsZ plays a central role at the earliest known step of septation. Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements. In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired. Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein. FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins. We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP. The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E. coli.

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.

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).

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.

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.

Structure and expression of the cell division genes ftsQ, ftsA and ftsZ

The essential cell division genes ftsQ, ftsA and ftsZ map in a cluster of cell envelope genes located at two minutes on the Escherichia coli genetic map and appear to constitute an atypical operon. These closely clustered genes are all transcribed in the same direction and yet each of these genes was independently cloned on a multicopy plasmid and found to be expressed. Tn5 insertion mutagenesis of this region confirmed that these genes were independently expressed, indicating that each of these genes has its own promoter. However, maximum expression of the distal ftsZ gene required the promoter for the proximal ftsQ gene. The proximal ftsZ promoter was located within the ftsA structural gene and the proximal ftsA promoter was located within the ftsQ structural gene. No transcription terminators were evident from the DNA sequence analysis of this region. The sequence analysis also revealed that the termination codon for ftsQ overlapped the initiation codon of the ftsA gene and that the ftsA and ftsZ genes were separated by 60 base-pairs. The ftsQ gene product has a calculated M r of 31,432 and the ftsA gene product has a calculated M r of 45,327. The structure and expression of these genes are discussed.