Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Abstract

20Bacterial cells are powerful models for understanding how cells divide and accomplish global 21 regulatory programs. In Caulobacter crescentus a cascade of essential master regulators regulate the 22 correct and sequential activation of DNA replication, cell division and development of different cell 23 types. Among them CtrA plays a crucial role coordinating all those functions. Despite decades of 24 investigation, no control by non-coding RNAs (ncRNAs) has been linked to Caulobacter cell cycle. 25Here, for the first time we describe the role of a novel essential factor named CcnA, a ncRNA located 26 at the origin of replication, activated by CtrA and responsible for the rapid and strong accumulation of 27 CtrA itself. In addition CcnA is also responsible for the inhibition of GcrA translation by direct 28 interaction with its UTR region. By a combination of probing experiments and mutagenesis, we 29 propose a new mechanism by liberation (CtrA) or sequestration (GcrA) of the Ribosome Binding Site 30 (RBS). CcnA role is conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, 31 representing indeed a conserved and fundamental process regulating cell cycle in Rhizobiales and32 Caulobacterales.73 responsible for the specific and highly regulated proteolysis of CtrA. 74In Caulobacter, the regulation of gene expression by ncRNAs has revealed few examples. Initially 75 only 27 ncRNAs were described in this organism (Landt et al., 2008). CrfA is an sRNA involved in 76 adaptation to carbon starvation (Landt et al., 2010). GsrN is involved in the response to σ T -dependent 77 multiple stresses (Tien et al., 2017). Finally ChvR has been recently characterized as a ncRNA that is 78 expressed in response to DNA damage, low pH, and growth in minimal medium (Fröhlich et al.,79 2018). However as new recent approaches using RNAseq and post-genomic techniques expanded the 80 plethora of ncRNA candidates to more than 100 (Zhou et al., 2015), predictions of their integration 81 into the cell cycle circuit (Beroual et al., 2018) has suggested that several new candidate ncRNAs 82 should be deeply studied. 83Here we investigated the role of a ncRNA, named CcnA, that belongs to the origin of replication of 84 Caulobacter chromosome. We studied its role by the construction of deletion mutants, silencing by 85 expression of its antisense and over expression. Results presented in this work identified the mRNAs 86 of CtrA and GcrA, two master regulators of cell cycle, as main targets of this ncRNA. Data were 87 88 closely related organism such as Sinorhizobium meliloti suggested its potential conservation across 89 bacteria. 91 Results 92CcnA expression is regulated by CtrA 94Based on previous results (Zhou et al., 2015) we observed that CCNA_R0094, here named Cell Cycle 95 non-coding RNA A (CcnA), expression peaks after few minutes from the accumulation of ctrA 96 transcript and protein, in the second half of the S-phase, when P2, the second ctrA promoter, is 97 activated (Fig 1A). 98We designed primers to dete...

Cold Regulation of Genes Encoding Ion Transport Systems in the Oligotrophic Bacterium Caulobacter crescentus

“…This, in turn, could inhibit the PTS Ntr relay, limiting (p)ppGpp and delaying the progression of the cell cycle ( 56 , 65 , 66 ). This is also in agreement with the upregulation of the cell-cycle-regulated sRNA CcnA, which also leads to a decrease in the growth rate ( 52 ).…”

“…Although the gene for the RNA chaperone Hfq was not differentially expressed, previous studies have shown that it participates in this type of regulation, acting as an intermediate between the sRNA and its target ( 50 , 51 ). Among the most upregulated sRNAs is the cell cycle-regulated CcnA (R0094) (11-fold), which is responsible for controlling two master regulators of the cell cycle, CtrA and GcrA ( 52 ). As the overexpression of CcnA leads to a slow growth phenotype ( 52 ), it is tempting to speculate that it contributes to the decrease in growth rates observed at low temperatures.…”

“…Among the most upregulated sRNAs is the cell cycle-regulated CcnA (R0094) (11-fold), which is responsible for controlling two master regulators of the cell cycle, CtrA and GcrA ( 52 ). As the overexpression of CcnA leads to a slow growth phenotype ( 52 ), it is tempting to speculate that it contributes to the decrease in growth rates observed at low temperatures.…”

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus

“…The heatmap figure was obtained with the R package Pheatmap and integrates the above annotations with gene expression data in the present work ( S12 Fig ). All fastQ files are available from the Dryad database (doi: 10.5061/dryad.4mw6m909k ) [ 88 ].…”