The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5’ Leader Featuring the sRNA UpsM

Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5’ UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5’ UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5’ UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5’ UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae

Northern blot analysis reveals Hfq and target mRNA dependent processing of UpsM by RNase E.

<p>(A) Detection of UpsM (206 nt) and the UpsM processing product (130 nt) by Northern blot analysis of TEX treated and untreated total RNA isolated from <i>R</i>. <i>sphaeroides</i> after 90 min ¹O₂ stress. (B) Left: Comparison of the UpsM processing pattern via Northern blot analysis of total RNA isolated from <i>R</i>. <i>sphaeroides</i> 2.4.1 (WT) to RNA isolated from a mutant strain expressing a thermosensitive RNase E variant from <i>E</i>. <i>coli</i> (<i>rne</i>^{<i>E</i>.<i>c</i>.ts}) after growth at 32, 37 or 42°C for 30 min or during ¹O₂ stress. Right: Comparison of the processing pattern via Northern blot analysis to strains lacking Hfq, RpoHI, RpoHII or RpoHI and RpoHII after 0 and 60 or 0 and 90 min of ¹O₂ stress. Signals of 5S rRNA serve as loading control.</p

Comprehensive view of <i>mraZ</i> upstream regions in different species.

<p>Terminator predictions are indicated in red. Respective energies are given in kcal/mol. Regions between Start and Stop codons in frame are shown as grey bars. Transcription start sites are derived from public available deep sequencing data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165694#pone.0165694.s004" target="_blank">S4 Fig</a> for details). The phylogenetic tree was build using clustalx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165694#pone.0165694.ref058" target="_blank">58</a>] (NJ, 10000 bootstraps) based on a clustalOmega [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165694#pone.0165694.ref059" target="_blank">59</a>] alignment of the respective <i>mraZ</i> coding regions. Bootstrap support values are indicated. Seemingly the long <i>mraZ</i> 5’ UTR with an intrinsic terminator is special to the family of <i>Rhodobacteraceae</i>.</p

dRNA-seq shows a long 5’ UTR of the <i>mraZ</i> gene in <i>R</i>. <i>sphaeroides</i>.

<p>Modified screenshots taken from IGB (integrated genome browser) visualizing the coverage at the genetic locus of <i>mraZ</i>. Shown are normalized cDNA reads on a large scale (upper two panels) and a smaller scale (lower two panels) obtained from TEX treated and untreated total RNA isolated from an exponentially and microaerobically grown <i>R</i>. <i>sphaeroides</i> 2.4.1 culture. The genetic context is displayed at the bottom. <i>mraZ</i> is the first gene of the <i>dcw</i> gene cluster. Position 1 reflects the TSS of sRNA UpsM (206 nt) 268 nucleotides upstream of <i>mraZ</i>. The terminator of UpsM is indicated as hairpin structure and a processing site within the sRNA is highlighted by an arrow.</p

Structural analysis of UpsM.

<p>Analogous structured regions are indicated as R1-R4. RNAfold structure of UpsM in <i>R</i>. <i>sphaeroides</i> with and without constraint terminator (R4).</p

Transcription of <i>mraZ</i> is enabled by the UpsM promotor.

<p>(A) β-galactosidase activity assays of <i>R</i>. <i>sphaeroides</i> 2.4.1 with reporter plasmids with <i>mraZ</i>::<i>lacZ</i> translational fusion and <i>mraZ</i> upstream regions of varying length (long upstream region including the promotor of UpsM, 188 and 67 upstream nucleotides). pPHU235 represents the empty vector control. For each strain, three independent biological experiments with technical duplicates were performed. Error bars indicate standard deviations and an asterisk a significance level of P<0.01 compared to pPHU235. (B) 5’ RACE with RNA from <i>R</i>. <i>sphaeroides</i> 2.4.1 after 90 min of ¹O₂ stress. cDNA was generated with the primer depicted as black arrow, whereas cDNAs were amplified by the primer shown as grey arrow. The PCR product was visualized on a gel (10% PAA/TBE) by ethidium bromide staining. 5’ ends (dashed lines) identified by subcloning and sequencing and their corresponding frequencies are highlighted. (C) qRT-PCR products of primer pairs pUpsM, pmraZ (155 bp and 153 bp, both specific for the corresponding mRNA segments) and pUpsM_<i>mraZ</i> (143 bp, spanning from UpsM to <i>mraZ</i>) visualized on a gel (10% PAA/TBE) by ethidium bromide staining. Samples without initial RT step were loaded as control. On the right relative transcript levels are shown in relation to the product quantity of primer pair pUpsM_<i>mraZ</i>. qRT-PCRs were performed in technical duplicates with RNA from three biological independent and unstressed <i>R</i>. <i>sphaeroides</i> 2.4.1 cultures. Error bars indicate standard deviations. (D) β-galactosidase activity assays of <i>R</i>. <i>sphaeroides</i> 2.4.1 conjugated with a reporter plasmid with translational <i>lacZ</i> fusion to the start codon (ATG) within the UpsM gene in comparison to the promoter-less empty vector control (pPHU235) and a control plasmid (pPHU4352) containing a strong 16S rRNA promoter. For each strain, three independent biological experiments with technical duplicates were performed. Error bars indicate standard deviations and an asterisk a significance level of P<0.01 compared to both controls.</p