Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start.

The dicA1 mutation, located in the replication termination region of Escherichia coli at 34.9 min, confers a temperature-sensitive, division defective phenotype to its hosts. Previous analysis had suggested that dicA codes for a repressor of a nearby division inhibition gene dicB. We show now that gene dicB is part of a complex operon. Five open reading frames (ORFs 1 to 5) preceeded by a promoter sensitive to dicA repression are found within a 1500 bp segment, and are organized into two clusters separated by a long untranslated region. Evidence for expression of these ORFs was obtained from in vitro or in vivo translation of plasmid-coded genes. IPTG-dependent cell filamentation was obtained when either the entire or the C-terminal part of the fourth ORF was placed under control of the lac promoter. In both cases, a 7 KD protein corresponding to translation from an in-frame ATG of ORF4 (dicB) was made. We propose that this C-terminal protein is the division inhibitor synthesized in dicA1 mutants.     

A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli

Summary Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome. The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions. Temperature-sensitivity conferred by mutation dicA1 in a recA backround was suppressed by the presence of hybrid plasmids carrying the wild-type gene. In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval. These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.     

Cell division inhibition gene dicB is regulated by a locus similar to lamboid bacteriophage immunity loci

Summary A mutation (dicA1) of a repressor gene located in the terminus region of the Escherichia coli chromosome has previously been shown to lead to temperature-dependent inhibition of division, and to be complemented by plasmids carrying either dicA or an adjacent gene dicC. In this study, operon fusions in the region coding for the division inhibition gene dicB have been used to show that temperature sensitivity does not result from high temperature inactivation of the dicA repressor. Sequence comparisons indicate that dicA and dicC are similar to genes c2 and cro respectively of bacteriophage P22, and carry similarly organized tandem operators, indicating a common evolutionary origin for dicAC and P22 immC. Nevertheless, the consensus half-operator sequence of dicAC, TGTTAGYYA, differs significantly from that of P22 immC (ATTTAAGAN). an analysis of the in vivo control of promoters dicAp, dicBp and dicCp placed upstream of malQ shows that the dicAC system is functionally similar to that of an immunity region, with the possible exception of an absence of pairwise cooperative binding. Our results also indicate that the dicA1 mutation causes a switch to permanent control by dicC at all temperatures.     

A cysteine-rich extracellular protein containing a PA14 domain mediates quorum sensing in Dictyostelium discoideum

Much remains to be understood about quorum-sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of discoidin-inducing complex (DIC), an ~400-kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing proteolytic digests. DicA1 and DicB were expressed in Escherichia coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression, while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 has a molecular mass of 80 kDa and has a 24-amino-acid cysteine-rich repeat that is similar to repeats in Dictyostelium proteins, such as the extracellular matrix protein ecmB/PstA, the prespore cell-inducing factor PSI, and the cyclic AMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum-sensing signal regulating discoidin gene expression during Dictyostelium growth and development.     

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.     

The E. coli divE mutation, which differentially inhibits synthesis of certain proteins, is in tRNASer1.

The temperature-sensitive divE mutant of Escherichia coli cannot synthesize certain membrane and cytoplasmic proteins at a non-permissive temperature. Growth of the mutant cells is arrested at a specific stage of the cell cycle when exposed to the non-permissive conditions, suggesting that the divE mutant possesses a defect in cell division control. From sequence determination of a cloned 1.35-kbp DNA fragment that complements the temperature-sensitive divE42 mutation, we characterized two genes in the segment ; one for tRNASer1 and the other for a 23 500 dalton protein. In parallel experiments we cloned the homologous 1.35-kbp DNA fragment from the divE42 mutant and determined its entire nucleotide sequence. Comparison of the two sequences showed that the mutation site is located not in the protein gene, but in the tRNA gene, where A10 is replaced by G10 in the D-stem. Lambda transducing phages carrying the subcloned tRNASer1 gene complemented the divE42 mutation, thereby confirming the conclusion obtained from sequence analyses of the fragments. This finding indicates that tRNASer1 is specifically involved in regulation of cell cycle-specific protein synthesis, coupled with an important step in the process of cell division, or that usage of serine tRNA is functionally specific for the biosynthesis of certain proteins.     

Identification of the ftsA gene product.

A nonsense mutation was identified in the essential cell division gene ftsA of Escherichia coli. A gamma-transducing phage was isolated which complemented this mutation. This phage programmed the synthesis of four bacterial proteins in UV-irradiated cells. By substituting the nonsense mutation for the ftsA+ allele in this transducing phage and comparing the proteins programmed by it in UV-treated Su+ and Su- cells, the product of the ftsA gene was identified as a protein with a molecular weight of 50,000.     

Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans

In A. nidulans, the temperature-sensitive cell cycle mutation bimC4 causes an elevated mitotic index at restrictive temperature. Under restrictive conditions the mutation interferes with separation of the spindle pole bodies, causes abnormal spindle morphology, and prevents nuclear division. We have cloned and sequenced the wild-type bimC gene. The predicted protein product has homology to Drosophila kinesin heavy chain. We conclude that this kinesin-like protein has an important role in nuclear division in Aspergillus.     

Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts).

A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation. Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed. The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division. The ftsN gene was located at 88.5 min on the E. coli genetic map just downstream of the cytR gene. ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death. DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da. The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins. The presumed extracellular domain was unusual in that it was rich in glutamine residues. A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.     

Analysis of glutathione peroxidase 1 gene polymorphism and Keshan disease in Heilongjiang Province, China

Keshan disease (KD) is an endemic cardiomyopathy associated with selenium deficiency. Recent studies indicate that glutathione peroxidase 1 (GPx1) mutation decreases GPx activity in myocardial cells and increases the risk of KD. To further clarify the correlation between GPx1 polymorphism and KD, we analyzed GPx1 polymorphism, blood selenium levels and GPx activity in KD patients and healthy controls in Heilongjiang Province. Four and 24 new mutation loci in the promoter and the exon region, respectively, of the GPx1 gene were found in the subjects, in contrast with the previously reported loci. There were no significant differences in the mutation frequency of these loci between the KD group and controls (chi-square test; P > 0.05). However, the mutation frequency of exon 474 was higher in the KD group (7/36) than in controls (2/41), and GPx activity was lower in the mutation group (90.475 ± 23.757 U/L) than in the non-mutation group (93.947 ± 17.463 U/L). Further investigation is necessary to clarify a possible causality between GPx1 exon 474 mutation and KD.     

A lacZ-ftsZ gene fusion is an analog of the cell division inhibitor sulA.

An in-frame lacZ-ftsZ gene fusion under lac control was fortuitously constructed by subcloning an EcoRI fragment that contains approximately 90% of the ftsZ gene. The identity of the gene fusion was confirmed by isolating an amber mutation in the hybrid gene and then using it to reconstruct the ftsZ gene, which now contained an amber mutation. The hybrid protein (ZZ), which does not possess ftsZ activity, contains seven amino acids of lacZ at its amino terminal end, followed by 35,000 daltons of the carboxyl end of the ftsZ protein. Induction of the hybrid protein resulted in a rapid cessation of cell division which could be reversed by removing the lac inducer. This inhibition of division could be prevented by an increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ, which is known to code for an altered but functional ftsZ protein. An increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ is known to overcome sulA-mediated inhibition of division during the SOS response. Thus, our results suggest that ZZ is an analog of sulA and may aid in determining how sulA inhibits cell division.     

Methionyl-transfer ribonucleic acid deficiency during G1 arrest of Saccharomyces cerevisiae.

The mesl- mutants of Saccharomyces cerevisiae cease division and accumulate in the G1 interval of the cell cycle when deprived of methionine or shifted from 23 to 36 degrees C in the presence of methionine. Synchronous cell cycle arrest results from a deficiency of charged methionyl-transfer ribonucleic acid (methionyl-tRNAMet) as shown by direct measurement of the in vivo pools of methionine, S-adenosylmethionine, and methionyl-tRNAMet. The deficiency of methionyl-tRNAMet in these cells is the consequence of a lesion in a single gene, mes1. mes1 appears to be the structural gene for the methionyl-tRNA synthetase because some revertants of this mutation exhibited a thermolabile methionyl-tRNA synthetase in vitro. A sufficient hypothesis to explain these and previous results is that the control of cell division by S. cerevisiae in response to nutrient limitation is mediated through aminoacyl-tRNA or subsequent steps in protein biosynthesis.     

Allosteric activation mechanism of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknB

The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 ? from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.     

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.     

Regulated arrest of cell proliferation mediated by yeast prt1 mutations

Several temperature-sensitive cell-division-cycle (cdc) mutations differentially affect the regulatory step for cell proliferation in the yeast. Saccharomyces cerevisiae, including one mutation termed cdc63-1, which resides in a previously known gene called PRT1. Other mutations in the PRT1 gene have been shown by others to affect an initiation step in protein synthesis. Here we show that at the appropriate nonpermissive temperature each prt1 mutation can produce a uniform and concerted arrest of cell division; the prt1-1 mutation, like cdc63-1, is shown to arrest cells specifically at the regulatory step for cell proliferation. This response of cessation of cell division is different from the response of cells to an equivalent limitation of protein synthesis using cycloheximide or verrucarin A, which implies that the PRT1 gene product could separately influence both cellular growth via protein synthesis and events in the regulation of cell proliferation.     

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.     

Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the orientation of the plane of cell division. We have cloned the rodC gene and have localized the site of the rodC1 lesion. To identify the rodC gene product, we have subjected several plasmid clones containing B. subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli. A 68 kiloDalton protein has been identified as the rodC gene product. This is the initial cloning of a cell division gene and the identification of its product from B. subtilis. The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid.