Regulatory RNAs in Bacteria

Regulation by Small RNAs in Bacteria: Expanding Frontiers

Cells are able to navigate environments, communicate, and build complex patterns by initiating gene expression in response to specific signals. Engineers need to harness this capability to program cells to perform tasks or build chemicals and materials that match the complexity seen in nature. This review describes new tools that aid the construction of genetic circuits. We show how circuit dynamics can be influenced by the choice of regulators and changed with expression “tuning knobs.” We collate the failure modes encountered when assembling circuits, quantify their impact on performance, and review mitigation efforts. Finally, we discuss the constraints that arise from operating within a living cell. Collectively, better tools, well-characterized parts, and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.

/pdf/principles-of-genetic-circuit-design-1stvh1u9g2.pdf

Principles of genetic circuit design

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45 degrees N to 40 degrees S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.

Ecological Genomics of Marine Picocyanobacteria

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5′ annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

https://www.pnas.org/content/pnas/108/5/2124.full.pdf

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

The ssr3341 locus was previously suggested to encode an orthologue of the RNA chaperone Hfq in the cyanobacterium Synechocystis sp. strain PCC 6803. Insertional inactivation of this gene resulted in a mutant that was not naturally transformable and exhibited a non-phototactic phenotype compared with the wild-type. The loss of motility was complemented by reintroduction of the wild-type gene, correlated with the re-establishment of type IV pili on the cell surface. Microarray analyses revealed a small set of genes with drastically reduced transcript levels in the knockout mutant compared with the wild-type cells. Among the most strongly affected genes, slr1667, slr1668, slr2015, slr2016 and slr2018 stood out, as they belong to two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein SYCRP1. This suggests a link between cAMP signalling, motility and possibly the involvement of RNA-based regulation. This is believed to be the first report demonstrating a functional role of an Hfq orthologue in cyanobacteria, establishing a new factor in the control of motility.

The cyanobacterial homologue of the RNA chaperone Hfq is essential for motility of Synechocystis sp PCC 6803

Small RNAs Establish Delays and Temporal Thresholds in Gene Expression

Background
The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.

/pdf/transcriptomic-response-to-prolonged-ethanol-production-in-21g319yaxw.pdf

Dennis Dienst

Papers

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

The cyanobacterial homologue of the RNA chaperone Hfq is essential for motility of Synechocystis sp PCC 6803

Small RNAs Establish Delays and Temporal Thresholds in Gene Expression

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803.