The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

Abstract

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

Dynamic translation regulation in Caulobacter cell cycle control

“…Caulobacter strain NA1000 was grown in minimal growth medium (M2G) (11) and synchronized by isolating swarmer cells (41,42). Six samples were taken at 30-min intervals over the span T = 5 to T = 150 min.…”

“…Thus, RPKM nt = N nt / (len n /R t ) where N nt = number of reads for gene n at time point t, len n = length of gene n in kilobases, and R t = total number of non-rRNA/non-tRNA reads in the sample taken at time t in millions). To eliminate data from initiating or terminating ribosomes, the first 10 and last 5 codons were excluded from the RPKM calculation (11,46). However, for genes with small (fewer than 50 codons) ORFs, the entire length of the ORF was included in the RPKM calculation.…”

“…The circular 4-Mb genome has 3,885 ORFs and 199 noncoding RNAs (5,6). mRNA profiling by microarrays or RNA-seq (7)(8)(9)(10) and global promoter activity profiles from 5′ Global RACE experiments (11) have shown that several hundred Caulobacter mRNAs have significant temporal transcriptional variation over the cell cycle. The expression of cell cycle-controlled mRNAs largely correlates with the times they are required for the functional modules that implement progression of the cell cycle (8).…”

Cell cycle progression inCaulobacterrequires a nucleoid-associated protein with high AT sequence recognition

“…Many cell cycle-regulated genes are subject to combinatorial control by two or more master regulators of cell cycle progression (14). To determine whether GapR binds promoters that are also occupied by known master regulators, we compared the GapR genome occupancy profile with that of transcriptional regulators known to modulate target gene expression in a cell cycle-dependent manner.…”

“…Each morphotype expresses a unique complement of genes that encode cell type-specific functions (e.g., motility, chemotaxis, encapsulation, or chromosome replication), the regulation of which can be attributed, at least in part, to an ensemble of interdependent, spatiotemporally restricted global transcriptional regulators that are organized into a highly robust control circuit (7)(8)(9)(10)(11)(12)(13)(14)(15). However, the control of more than 40% of cell cycle-regulated promoters cannot be accounted for by these master regulators (14), implying the existence of additional regulatory proteins that influence development through interaction(s) with the Caulobacter genome. Such factors could include NAPs, which can directly or indirectly modulate gene regulation (1).…”