Caulobacter crescentus represents a remarkable model system to investigate global regulatory programs in bacteria. In particular, several decades of intensive study have revealed that its cell cycle is controlled by a cascade of master regulators, such as DnaA, GcrA, CcrM, and CtrA, that are responsible for the activation of functions required to progress through DNA replication, cell division and morphogenesis of polar structures (flagellum and stalk). In order to accomplish this task, several post-translational (phosphorylation and proteolysis) and transcriptional mechanisms are involved. Surprisingly, the role of non-coding RNAs (ncRNAs) in regulating the cell cycle has not been investigated. Here we describe a bioinformatic analysis that revealed that ncRNAs may well play a crucial role regulating cell cycle in C. crescentus. We used available prediction tools to understand which target genes may be regulated by ncRNAs in this bacterium. Furthermore, we predicted whether ncRNAs with a cell cycle regulated expression profile may be directly regulated by DnaA, GcrA, and CtrA, at the onset, during or end of the S-phase/swarmer cell, or if any of them has CcrM methylation sites in the promoter region. Our analysis suggests the existence of a potentially very important network of ncRNAs regulated by or regulating well-known cell cycle genes in C. crescentus. Our hypothesis is that ncRNAs are intimately connected to the known regulatory network, playing a crucial modulatory role in cell cycle progression.

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle–regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5′ untranslated region (5′ UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

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...

Summary The bacterial envelope is a remarkable and complex compartment of the prokaryotic cell in which many essential functions take place. The article by Herrou and collaborators (Herrou et al., in press), by a clever combination of structural analysis, genetics and functional characterization in free‐living bacterial cells and during infection in animal models, elucidates a new factor, named EipA, that plays a major role in Brucella spp envelope biogenesis and cell division. The authors demonstrate a genetic connection between eipA and lipopolysaccharide synthesis, specifically genes involved in the synthesis of the O‐antigen portion of lipopolysaccharide (LPS). Beyond its crucial role in Brucella physiology, the conservation of EipA in the class Alphaproteobacteria urges microbiologists to pursue future investigation of its homologs in other species belonging to this important group of bacteria.