Synthesis of Bacterial Flagella II. PBSl Transduction of Flagella-specific Markers in Bacillus subtilis

The linkage relationship of mutants involved in the synthesis of flagella was determined by PBSl transduction. Mutants that affect the structure of flagellin (hag) and temperature-sensitive mutants (flaTS) that produce flagella when grown at 37 C but not when grown at 46 C were examined. All of the mutants were found to be linked to the hisA1 marker. The flaTS mutants fell into three clusters. Group A contained the majority of mutants which were loosely grouped around the hag locus. Group B mutants were segregated from the hag locus and appeared closely linked to the phage adsorption site gene (gtaA), and group C was only loosely linked to hisA1 and thus far contains only one mutant. A flagella locus (ifm) affecting both the degree of motility and level of flagellation was shown to map near group A. Mutants affecting motility (mot) were not linked to hisA1 by PBSl transduction. Several markers previously shown to link to hisA1 were ordered with respect to hisA1 and the flagellar genes.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation.

F plasmid replication during the Escherichia coli division cycle was investigated by using the membrane-elution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specific. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome--initiation occurred when a constant mass per origin was achieved--except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.     

Control of membrane protein synthesis in bacillus subtilis

In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation. In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle. Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles.I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis.     

Relationship between cellular autolytic activity, peptidoglycan synthesis, septation, and the cell cycle in synchronized populations of Streptococcus faecium.

Synchronized, slowly growing (TD = 70 to 80 min) cultures were used to study several wall-associated parameters during the cell cycle: rate of peptidoglycan synthesis, septation, and cellular autolytic activity. The rate of peptidoglycan synthesis per cell declined during most of the period of chromosome replication (C), but increased during the latter part of C and into the period between chromosome termination and cell division (D). An increase in cellular septation was correlated with the increased rate of peptidoglycan synthesis. Cellular autolytic capacity increased during the early portion of C, reached a maximum late in C or early in D, and declined during D. Inhibition of DNA synthesis during C prevented the decline in autolytic capacity at the end of the cell cycle, caused a slight reduction in the rate of peptidoglycan synthesis, delayed but did not prevent septation, and prevented the impending cell division by inhibiting cell separation. Inhibition of DNA synthesis during D did not prevent the increase in autolytic capacity during the next C phase, but, once again, prevented the decline at the end of the subsequent cycle. Thus, increased autolytic capacity at the beginning of the cell cycle did not seem to be related to chromosome initiation, whereas decreased autolytic capacity at the end of the cell cycle seemed to be related to chromosome termination. The data presented are consistent with the role of autolytic enzyme activity in the previously proposed model for cell division of S. faecium (G.D. Shockman et al., Ann. N.Y Acad. Sci. 235:161-197, 1974).     

Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.     

Comparison among patterns of macromolecular synthesis in Escherichia coli B/r at growth rates of less and more than one doubling per hour at 37 degrees C.

In Escherichia coli B/r, the relationship between the patterns of chromosome replication and of synthesis of envelope components differs at various growth rates. At growth rates greater than 1.0 doubling per h at 37 degrees C, the average mass and age at initiation of rounds of chromosome replication are similar to those at increase in incorporation of precursors into a major outer membrane protein and phosphatidylethanolamine. At growth rates less than 1.0 doubling per h at 37 degrees C the average mass and age at increase in the synthesis of these envelope components differ from those at initiation of chromosome replication. The average cell mass per chromosomal origin at initiation of rounds of chromosome replication is not a constant and varies between growth rates greater and less than 1.0 doubling per h.     

Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome.     

Synthesis of photoreactivating enzyme in synchronous and asynchronous cultures of Escherichia coli

The technique of flash photolysis was used to study cellular variations in the number of photoreactivating enzyme (PRE) molecules during the cell division cycle of the UV-sensitive E. coli strain B_S?1. No variations in the number of PRE molecules per genome were observed throughout the cell division cycle when synchronized cells cultured in either glucose-minimal or succinate-minimal medium were used. This is interpreted to mean that PRE synthesis is continuous throughout the cell cycle for glucose-grown cells, but may stop at the time chromosome replication ceases prior to division, in succinate-grown cells. The effect of growth rate and stage of growth on cellular PRE content in asynchronous cultures was also determined. Variations in the number of PRE per genome were observed for both synchronous and asynchronous cells cultured in different media and occurred in a manner that suggested a dependence on growth rate. PRE per genome increased with generation time. Stationary phase cells from each culture medium (nutrient broth, glucose-minimal, succinate-minimal) had more PRE per genome than did respective log phase cells. It is suggested that PRE synthesis may be controlled by some aspect of chromosome replication.     

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants

The genetic aspects of ori C replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dna A cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dna A integrated at the att B locus in dna A null background was exchanged with an incoming plasmid bearing a mutagenized dna A gene. Next, the mutants that were defective for growth at 30°C, a non-permissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dna A gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dna A expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dna Acos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dna A promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Chromosome Replication and Cell Division in Escherichia coli at Various Temperatures of Growth

The effect of temperature on the growth rate and the pattern of chromosome replication during the division cycle of Escherichia coli B/r growing in various media was investigated. The time between divisions, the time for a round of replication (C), and the time between completion of a round and cell division (D) were threefold longer at 21 C than at 37 C. At all temperatures and in all media, D equalled one-half C, suggesting that a common mechanism controls chromosome replication and the progression of the cell toward division after completion of a round of replication.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

An easy-to-use simulation program demonstrates variations in bacterial cell cycle parameters depending on medium and temperature

Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations. Here, a Visual Basic based simulation program was written for the computation of theoretical DNA distributions for different choices of cell cycle parameters (C and D phase durations, doubling time etc). These cell cycle parameters can be iterated until the best fit between the experimental and theoretical DNA histograms is obtained. The Excel file containing the simulation software is attached as supporting information.Cultures of Escherichia coli were grown at twelve different media and temperature conditions, with following measurements by flow cytometry and simulation of the DNA distributions. A good fit was found for each growth condition by use of our simulation program. The resulting cell cycle parameters displayed clear inter-media differences in replication patterns, but indicated a high degree of temperature independence for each medium. The exception was the poorest medium (acetate), where the cells grew with overlapping replication cycles at 42 °C, but without at the lower temperatures.We have developed an easy-to-use tool for determination of bacteria's cell cycle parameters, and consequently the cells' chromosome configurations. The procedure only requires DNA distribution measurements by flow cytometry. Use of this simulation program for E. coli cultures shows that even cells growing quite slowly can have overlapping replication cycles. It is therefore always important not only to assume cells' replication patterns, but to actually determine the cell cycle parameters when changing growth conditions.