Changes of ploidy during the Azotobacter vinelandii growth cycle.

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory.     

Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes.

The properties of Azotobacter vinelandii deoxyribonucleic acid (DNA) and folded chromosomes were studied and compared to those of Escherichia coli as a standard. Based on melting temperature and buoyant density measurements, the guanosine + cytosine content of purified A. vinelandii DNA was 65%, whereas that of E. coli DNA was 50%. The results of renaturation studies showed that the unique DNA sequence lengths of the two organisms were similar with Cot1/2 values of 7.3 +/- 0.4 mol.s/liter and 7.5 +/- 0.3 mol.s/liter, respectively, for A. vinelandii and E. coli. Folded chromosomes of A. vinelandii sedimented in a centrifugal field at a rate identical to those derived from E. coli, 1,600 to 1,700S. Based on the DNA content per cell and the mass of a single genome, A. vinelandii contains at least 40 chromosomes per cell.     

Multiple chromosomes of Azotobacter vinelandii.

The number of copies of the genes leuB, nifH, nifD, and nifK per cell of Azotobacter vinelandii has been determined to be about 80. A beta-lactamase gene was integrated into the A. vinelandii chromosome by single-point crossover. Subsequently, we have been able to detect nearly 80 copies of this beta-lactamase gene per cell of A. vinelandii when cultured for a large number of generations in the presence of ampicillin. The multiple copies of the beta-lactamase gene do not seem to be present on a single chromosome, as evident from the fragment obtained by digestion of cellular DNA with the appropriate restriction endonuclease. The kinetics of renaturation of DNA of A. vinelandii is suggestive of complexity similar to that of Escherichia coli. The DNA content of A. vinelandii, however, is 40 times that of E. coli. All these indicate the presence of multiple chromosomes, possibly as many as 80, in A. vinelandii.     

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Background

In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.

Results

We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.

Conclusions

We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.     

Physiological Studies of Encystment in Azotobacter vinelandii

Azotobacter vinelandii, in late exponential growth phase, encysts when the glucose in the medium is replaced with beta-hydroxybutyrate. A final cell division then occurs without apparent deoxyribonucleic acid (DNA) synthesis, resulting in a reduction from two to one nucleoids per cell and a final DNA content of 3.2 x 10(-14) g per cell. This is also the DNA content per cyst. A beta-hydroxybutyrate dehydrogenase is derepressed by the addition of the inducer and is identical to the enzyme in acetate-grown cells in its pH optimum, Michaelis constant for substrate, temperature-activity response, and mobility during electrophoresis in acrylamide gel.     

DNA and protein content relationship in the cells of mature cotyledons ofPisum sativum

Summary The relationship between the DNA and reserve protein contents have been investigated in the cells of mature cotyledons ofPisum sativum. The study was made on two seeds chosen, on preliminary analysis, as having a markedly different protein content per cotyledon. The seed with the higher protein content was of theCameor variety, the other was of theAmino variety. The DNA content per cell was measured with a scanning microspectrophotometer and the protein content per cell using morphometric methods. In both seeds, nuclei of the storage parenchyma were polyploid in a range from 8 to 64 C levels, but the average DNA content per cell was higher in the Cameor specimen. The protein content also, whether expressed per unit of protoplasm or per cell, was always higher in the Cameor seed. The greater protein content of this seed was due to a higher content of protein per cell. In both seeds the level of ploidy and the reserve protein content per cell increased in the same way from the peripheral zone to the centre of the cotyledon. A high positive correlation was found for the two seeds between the DNA content and the protein content per cell.     

Generating Autotetraploid Sporophytes and Their Use in Analyzing Mutations Affecting Gametophyte Development in the Fern Ceratopteris

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.     

Segregation studies in CHO hybrid cells: I. Spontaneous and mutagen-induced segregation events of two recessive drug-resistant loci

The process of segreation or phenotypic expression of two recessive drug-resistant loci from heterozygous Chinese hamster ovary hybrid lines is examined. The spontaneous segregation rates of phytohaemagglutinin resistance (Phar) and a temperature-dependent 8-azaguanine-resistant locus (Azarts) from heterozygous quasitetraploid lines using Luria-Delbruck fluctuation analysis were 5 X 10(-5) and 10(-5) events/cell/generation, respectively. In quasihexaploid lines, the latter rates increased 40- and 200-fold, respectively, and were dependent on the number of presumptive drug-sensitive allelel. The mutagens EMS, MNNG, ICR-170, ICR-191, and gamma rays significantly increased the frequency of segregation events. The mutagen-induced frequency of dominant mutations to ouabain (Ouar) and alpha-amanitin (Amar) rsistance in the same hybrid line was much lower in comparison to segregation events and was mutagen specific. The chromosome number per metaphase cell was more variable than DNA content in quasitetraploid lines. These properties of marker segregation are consistent with mechanisms of either restricted chromosome loss, rearrangement, or mutation.     

Alternative Function of the Electron Transport System in Azotobacter vinelandii: Removal of Excess Reductant by the Cytochrome d Pathway

The N(inf2)-fixing bacterium Azotobacter vinelandii was grown in an O(inf2)-regulated chemostat with glucose or galactose as substrate. Increasing the O(inf2) partial pressure resulted in identical synthesis of the noncoupled cytochrome d terminal oxidase, which is consistent with the hypothesis that A. vinelandii uses high rates of respiration to protect the nitrogenase from oxygen. However, cell growth on glucose showed a lower yield of biomass, higher glycolytic rate, higher respiratory rate, and lower cytochrome o content than cell growth on galactose. Elemental analysis indicated no appreciable change in the C-to-N ratio of cell cultures, suggesting that the major composition of the cell was not influenced by the carbon source. A poor coordination of glucose and nitrogen metabolisms in A. vinelandii was suggested. The rapid hydrolysis of glucose resulted in carbonaceous accumulation in cells. Thus, Azotobacter species must induce a futile electron transport to protect cells from the high rates of glucose uptake and glycolysis.     

Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air.

The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.     

Ectopic expression of the Suppressor of Underreplication gene inhibits endocycles but not the mitotic cell cycle in Drosophila melanogaster

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.     

Systematic segregation to mutant mitochondrial DNA and accompanying loss of mitochondrial DNA in human NT2 teratocarcinoma Cybrids

In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human rho(0) teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.     

Plasmid transformation of Azotobacter vinelandii OP

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.     

Kinetics of endomitosis in primary murine megakaryocytes

Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.     

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.     

Chromosome segregation control by Escherichia coli ObgE GTPase

Escherichia coli cells depleted of the conserved GTPase, ObgE, show early chromosome-partitioning defects and accumulate replicated chromosomes in which the terminus regions are colocalized. Cells lacking ObgE continue to initiate replication, with a normal ratio of the origin to terminus. Localization of the SeqA DNA binding protein, normally seen as punctate foci, however, was disturbed. Depletion of ObgE also results in cell filamentation, with polyploid DNA content. Depletion of ObgE did not cause lethality, and cells recovered fully after expression of ObgE was restored. We propose a model in which ObgE is required to license chromosome segregation and subsequent cell cycle events.     

Overexpression of the Tn5 transposase in Escherichia coli results in filamentation, aberrant nucleoid segregation, and cell death: analysis of E. coli and transposase suppressor mutations.

Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli. This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp. Instead, it was strictly correlated with the presence of a wild-type N terminus. Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect. This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation. Four E. coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci. These suppressor mutations map near essential genes involved in cell division and DNA segregation. One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min. A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E. coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell. Furthermore, the N terminus of Tnp is necessary for this interaction. The possible significance of this phenomenon for the transposition process is discussed.     

DNA segregation in Escherichia coli cells with 5-bromodeoxyuridine-substituted nucleoids.

The pattern of segregation of DNA in Escherichia coli K-12 was analyzed by labeling replicating DNA with 5-bromodeoxyuridine followed by differential staining of nucleoids. Three types of visible arrangement were found in four-nucleoid groups derived from a native nucleoid after two replication rounds. Type A, segregation of both old strands toward cell poles, appeared with the highest frequency (0.6 to 0.8). Type B, segregation of one old strand toward the cell pole and the other toward the cell center, was twice as frequent as type C, segregation of both old strands toward the cell center. These results confirm previous data showing that DNA segregation in E. coli is nonrandom while presenting a certain degree of randomness. The proportions of the three indicated types of arrangement suggest a new probabilistic model to explain the observed segregation pattern. It is proposed that DNA strands segregate either nonrandomly, with a probability of between 0 and 1, or randomly. In nonrandom segregation, both old strands are always directed toward cell poles. Experimental data reported here or by other authors fit better with the predictions of this model than with those of other previously proposed proposed deterministic or probabilistic models.