Genetic and genomic analysis of RNases in model cyanobacteria

Abstract

Cyanobacteria are diverse photosynthetic microbes with the ability to convert CO2 into useful products. However, metabolic engineering of cyanobacteria remains challenging because of the limited resources for modifying the expression of endogenous and exogenous biochemical pathways. Fine-tuned control of protein production will be critical to optimize the biological conversion of CO2 into desirable molecules. Messenger RNA (mRNA) are labile intermediates that play critical roles in determining the translation rate and steady state protein concentrations in the cell. The majority of studies on mRNA turnover have focused on the model heterotrophic bacteria Escherichia coli and Bacillus subtilis. These studies have elucidated many RNA modifying and processing enzymes and have highlighted the differences between these Gram-negative and Gram-positive bacteria, respectively. In contrast, much less is known about mRNA turnover in cyanobacteria. We generated a compendium of the major ribonucleases (RNases) and provide an in-depth analysis of RNase III-like enzymes in commonly studied and diverse cyanobacteria. Furthermore, using targeted gene deletion, we genetically dissected the RNases in Synechococcus sp. PCC 7002, one of the fastest growing and industrially attractive cyanobacterial strains. We found that all three cyanobacterial homologs of RNase III and a member of the RNase II/R family are not essential under standard laboratory conditions, while homologs of RNase E/G, RNase J1/J2, PNPase, and a different member of the RNase II/R family appear to be essential for growth. This work will enhance our understanding of native control of gene expression and will facilitate the development of an RNA-based toolkit for metabolic engineering in cyanobacteria.

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803

“…Data are based on two biological replicates. accumulation, possibly similar in relevance to RNase E in cyanobacteria [56]. Overall, the data suggest that CrhR seemingly performs roles that affect a limited set of the mRNA and ncRNA repertoire in Synechocystis.…”

Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002

“…All strains used in this study are listed in Supplemental Table S1 . A detailed description of the creation of the RNase III mutant cyanobacterial strains is outlined in ( 13 ), but in summary, antibiotic resistance markers flanked by 500 bp homology arms were introduced via natural transformation. After passaging on the appropriate antibiotic(s), the segregation of the strains was verified.…”

“…strain PCC 7002 (hereafter PCC 7002), for both its industrial applications in chemical production and its unique array of RNases. PCC 7002 contains both a homolog of RNase E (A0788) and RNase J (A1273) as well as three homologs of RNase III (A0061, A2542, A0384); the first two being full length RNase III’s while the third being a Mini-RNase III that lacks the double-stranded binding domain ( 13 ). These three RNase III homologs are not essential under standard growth conditions and have been deleted in all combinations ( 13 ).…”

“…PCC 7002 contains both a homolog of RNase E (A0788) and RNase J (A1273) as well as three homologs of RNase III (A0061, A2542, A0384); the first two being full length RNase III’s while the third being a Mini-RNase III that lacks the double-stranded binding domain ( 13 ). These three RNase III homologs are not essential under standard growth conditions and have been deleted in all combinations ( 13 ). Here, we aimed to elucidate the function of the three homologs of RNase III to determine if they played distinct or redundant roles in transcript turnover, what structures and sequences they target, and what genes and cellular activities are affected by their action.…”

De novodesign of heat-repressible RNA thermosensors inE. coli

“…Second, RNase E is native to E. coli , alleviating the need for the expression of a heterologous protein. Finally, both RNase E and its homologue RNase G are common in β- and γ-proteobacteria ( 25 ) as well as cyanobacteria ( 26 ), and about half of all eubacteria outside of these groups have at least one of these enzymes on its chromosome ( 25 ). This provides reason to believe that implementation of these thermosensors in other organisms is possible with host-specific optimization.…”