Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus.

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.

Regulation of stalk elongation by phosphate in Caulobacter crescentus.

In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli. In E. coli, pst mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both pstS and phoB mutants were deficient in phosphate transport. When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of phoB and pst mutants were opposite. phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media. Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB. In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB.

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus.

During exponential growth, each cell cycle of the alpha-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6-8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10(9) cfu ml(-1) to a minimum of 3x10(7) cfu ml(-1) after which cells gradually adopt an elongated helical morphology. For days 9-12, the cfu of the culture increase and stabilize around 2 x 10(8) cfu ml(-1). The viable cells have an elongated helical morphology with no constrictions and an average length of 20 microm, which is 15-20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.

Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA.

The cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N-terminal region of approximately 320 amino acids; a variable spacer region; and a conserved C-terminal region of eight amino acids. We show that FtsZ mutants missing different C-terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C-terminal conserved region, the linker, and 40 amino acids from the end of the N-terminal conserved region (FtsZdeltaC281) prevents the localization or the polymerization of FtsZ. Because two-hybrid analysis indicates that FtsZdeltaC281 does not interact with FtsZ, we hypothesize that FtsZdeltaC281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C-terminus of FtsZ (FtsZdeltaC485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C-terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.

Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.

Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy = pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy >> butoxy > propoxy = methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- = pentoxy- = propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and beta-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between beta-NF-type and DEX-type inducers may best be made using 7-methoxyphenoxazone as a substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatic CYP1A1 induction in rainbow trout by continuous flowthrough exposure to beta-naphthoflavone.

In order to assess the usefulness of CYP1A1 mRNA measurement as an environmental biomarker it was necessary to determine if hepatic P450 CYP1A1 mRNA induction is sustained during constant exposure to hepatic monooxygenase inducers. To accomplish this, rainbow trout (Oncorhynchus mykiss) were exposed, under flowthrough conditions, to beta-naphthoflavone (beta-NF), a known CYP1A1 inducer in fish. Trout were exposed to a beta-NF concentration of 1.0 mg beta-NF/liter, using dimethylformamide as carrier, for 1, 2, 4, and 8 days, followed by depuration in clean water for 1, 8, 14, and 35 days. In a second experiment, trout were exposed to beta-NF concentrations of 0.05, 0.10, and 0.50 mg beta-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, and 14 days, followed by depuration for 7 and 28 days. At the 1.0-mg beta-NF/liter concentration, ethoxyresorufin-O-deethylase (EROD) activity was significantly decreased by 4 days of exposure when compared to controls. At beta-NF concentrations of 0.05 to 0.50 mg beta-NF/liter EROD activity was increased compared to controls but was inversely related to the beta-NF concentration. Hybridizable CYP1A1 mRNA was increased approximately 40-fold over control levels at concentrations of 0.05 to 0.50 mg beta-NF/liter for 1, 3, and 7 days of exposure. In a third experiment, trout exposed to 0.05 mg beta-NF/liter for 2, 6, 12, 24, 32, 40, and 48 hr had increased (45- to 167-fold) EROD activity by 18 and 48 hr, respectively. Immunoreactive CYP1A1 protein was increased 46-fold at 48 hr and CYP1A1 mRNA was increased 29-fold at 48 hr of continuous beta-NF exposure. This is in contrast to previous experiments using intraperitoneal injection of beta-NF in which the induced CYP1A1 mRNA decreased to near control levels by 48 hr after injection. These data indicate that both CYP1A1 catalytic activity and immunoreactive protein are decreased at high inducer concentrations while mRNA levels remain elevated and continue to increase over time during continuous exposure. In a fourth experiment trout were continuously exposed to concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0 micrograms beta-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, 14, and 21 days, followed by clean water depuration for 1, 3, 7, 14, and 21 days EROD activity was significantly increased in a concentration-dependent manner over control by Day 1 of exposure with concentrations of 2.5, 5.0, and 10.0 micrograms beta-NF/liter.(ABSTRACT TRUNCATED AT 400 WORDS)