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Genome-wide identification and characterization of small RNAs in Rhodobacter capsulatus and identification of small RNAs affected by loss of the response regulator CtrA

ABSTRACT: We present the first genome-wide identification and characterizaion of 422 novel sRNAs in R. capsulatus. In addition we report a comparative analysis of conserved sRNAs across 24 bacterial species.

ORGANISM(S): Rhodobacter capsulatus

PROVIDER: GSE82056 | GEO | 2017/04/05

SECONDARY ACCESSION(S): PRJNA323840

REPOSITORIES: GEO

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Genome-wide identification and characterization of small RNAs in Rhodobacter capsulatus and identification of small RNAs affected by loss of the response regulator CtrA.

Project description:Small non-coding RNAs (sRNAs) are involved in the control of numerous cellular processes through various regulatory mechanisms, and in the past decade many studies have identified sRNAs in a multitude of bacterial species using RNA sequencing (RNA-seq). Here, we present the first genome-wide analysis of sRNA sequencing data in Rhodobacter capsulatus, a purple nonsulfur photosynthetic alphaproteobacterium. Using a recently developed bioinformatics approach, sRNA-Detect, we detected 422 putative sRNAs from R. capsulatus RNA-seq data. Based on their sequence similarity to sRNAs in a sRNA collection, consisting of published putative sRNAs from 23 additional bacterial species, and RNA databases, the sequences of 124 putative sRNAs were conserved in at least one other bacterial species; and, 19 putative sRNAs were assigned a predicted function. We bioinformatically characterized all putative sRNAs and applied machine learning approaches to calculate the probability of a nucleotide sequence to be a bona fide sRNA. The resulting quantitative model was able to correctly classify 95.2% of sequences in a validation set. We found that putative cis-targets for antisense and partially overlapping sRNAs were enriched with protein-coding genes involved in primary metabolic processes, photosynthesis, compound binding, and with genes forming part of macromolecular complexes. We performed differential expression analysis to compare the wild type strain to a mutant lacking the response regulator CtrA, an important regulator of gene expression in R. capsulatus, and identified 18 putative sRNAs with differing levels in the two strains. Finally, we validated the existence and expression patterns of four novel sRNAs by Northern blot analysis.

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Project description:The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase on R. capsulatus ctrA gene expression. These experiments indicate that ctrA expression is higher when cultures are grown in phototrophic (anaerobic) conditions compared with chemotrophic (aerobic) conditions, and is higher when grown in a minimal medium compared with a rich medium. We used several mutants to investigate possible regulatory pathways, and found that in R. capsulatus ctrA is not autoregulated but is regulated by a quorum-sensing system. The expression of ctrA increased as cell cultures moved through exponential phase and into stationary phase, with high levels of expression persisting long after culture turbidity plateaued. Although this growth phase-dependent pattern of expression was also observed in a quorum-sensing mutant, the magnitude of ctrA expression was about 50% of the wild-type strain at all phases. Furthermore, reduction of phosphate concentration in the growth medium decreased ctrA expression in a culture density-independent manner, whereas reduction of malic acid (carbon source) or ammonium (nitrogen source) concentration had no effect. The regulation of ctrA expression in R. capsulatus appears to require the coordination of multiple pathways involved in detecting a variety of environmental conditions.

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