Cell cycle expression and transcriptional regulation of DNA topoisomerase IV genes in caulobacter

Abstract

DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897-910, 1997). We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of parE and parC expression is not strictly coordinated, however. The rate of parE transcription increases ca. 20-fold during the G1-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through parE expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor sigma73. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the -10 and -35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication.

FIG. 1

Transcriptional regulation of parEp in synchronous swarmer and stalked cell cultures. (A) Transcriptional activity of a parEp-lacZ fusion (PC4828) during the C. crescentus swarmer cell cycle as assayed by radioimmune precipitation of β-galactosidase. Division occurred at 150 min. (B) Transcriptional activity of a parEp-lacZ fusion (PC4828) during the C. crescentus stalked cell cycle as assayed by radioimmune precipitation of β-galactosidase. Division occurred at 120 min. Activity is plotted as a percentage of maximal expression. Note that 1.0 division unit for the stalked cell cycle corresponds approximately to the period of 0.4 to 1.0 division unit of the swarmer cell cycle. Expression of pulse-labeled FlgE protein served as an internal control for these synchrony experiments and those shown in Fig. 2. The times of swarmer to stalked cell differentiation and completion of cell division (1.0 division unit) are indicated at the bottoms of panels A and B along with the corresponding G₁, S, and G₂ phases.

FIG. 2

Transcriptional regulation of parCp in synchronous swarmer and stalked cell cultures. (A) Transcriptional activity of a parCp-lacZ fusion (PC4480) during the swarmer cell cycle as assayed by radioimmune precipitation of β-galactosidase. Division occurred at 120 min. (B) Transcriptional activity of a parCp-lacZ fusion (PC4480) during the stalked cell cycle as assayed by radioimmune precipitation of β-galactosidase. Division occurred at 90 min, which corresponds to 1.0 division unit. Activity is plotted as a percentage of maximal expression. The cell cycle periods are as described in the legend for Fig. 1 and the text.

FIG. 3

Levels of ParE and ParC in synchronous cell cultures. (A) The 80-kDa ParE protein was detected by Western analysis of samples from a synchronous culture of strain CB15F at the times indicated. The last three lanes contain lysates of wild-type strain CB15, strain PC8830[divC307(Ts)], and strain PC4885[divC307(Ts), sueA020(Cs)], a spontaneous revertant of the divC307(Ts) strain grown at the restrictive temperature of 37°C. The level of the ParE protein was reduced in the divC307(Ts) strain and increased in the sueA020(Cs) strain relative to wild-type levels. Levels of ParE and ParC were quantified as described in Materials and Methods, and the level in swarmer cells at 0 min was normalized to 1.0 U. These measurements showed that the level of ParE increased from 1.0 U at 0 min to 2.1 U immediately before cell division at 0.8 to 0.9 division unit and then decreased after cell division to 1.6 U at 1.2 division units. Similar results were obtained when ¹²⁵I-labeled secondary antibodies were used; ParE levels determined in this assay (see Materials and Methods) increased from 1.0 U at 0 min to 1.9 U before division at 0.8 to 0.9 division unit and then decreased to 1.3 U after division at 1.2 division units. (B) The 87-kDa ParC protein was detected by Western analysis of lysates from a synchronous cell culture of strain CB15F. Assays of lysates of wild-type strains CB15 and PC8861[divF310(Ts)] grown at the restrictive temperature of 37°C are shown in the last two lanes. The level of the ParC protein was diminished in strain PC8861. Bands were quantified as described in Materials and Methods, but reproducible changes in ParC levels during the cell cycle were not detected.

FIG. 4

Identification of transcription start sites for the parE and parC genes. (A) S1 nuclease protection of the parE transcript. Lanes: 1, probe plus 100 μg of CB15 RNA; 2, probe plus 100 μg of CB15 RNA plus S1 nuclease; 3, probe plus 100 μg of yeast tRNA plus S1 nuclease. The transcription start site is indicated. (B) S1 nuclease protection of the parC transcript. Lanes: 1, probe plus 100 μg of CB15 RNA; 2, probe plus 100 μg of CB15 RNA plus S1 nuclease; 3, probe plus 100 μg of yeast tRNA plus S1 nuclease. The transcription start sites are indicated.

FIG. 5

Sequence of parE and parC promoter regions and fusion junctions to the lacZ reporter gene. (A) Nucleotide sequence of the parE gene promoter region. (B) Nucleotide sequence of the parC gene promoter region. The 5′ ends of promoter deletion constructs are indicated by arrows followed, in parentheses, by their position relative to +1, and the promoter activity relative to the activity of the full-length fragment (100 U) (see text and Materials and Methods). An asterisk indicates transcription initiation sites. Restriction sites, −35 and −10 sequences, translation stop codons and transcription initiation sites are underlined. Transcription and translation initiation sequences are in boldface. The translated ParE and ParC sequences are given above the nucleotide sequence. The “…” represents a gap in the presented sequence, and is followed by the sequence of the transcriptional fusion junction to the lacZ reporter construct.

FIG. 6

Alignment of parE and parC gene promoters with other C. crescentus gene promoters and to the ς⁷³ consensus recognition sequence (Fig. 6) (19). Bases matching the consensus sequence are underlined. N = any base; S = C/G; W = A/T; Y = C/T. The references for promoter sequences and transcriptional start sites are as follows: dnaA (45), dnaN and dnaX (36, 42), dnaK (1a), and gyrB (35, 36).