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Antisense RNA asPcrL regulates expression of photosynthesis genes in Rhodobacter sphaeroides by promoting RNase III-dependent turn-over of puf mRNA.

ABSTRACT: Anoxygenic photosynthesis is an important pathway for Rhodobacter sphaeroides to produce ATP under oxygen-limiting conditions. The expression of its photosynthesis genes is tightly regulated at transcriptional and post-transcriptional levels in response to light and oxygen signals, to avoid photooxidative stress by the simultaneous presence of pigments, light and oxygen. The puf operon encodes pigment-binding proteins of the light-harvesting complex I (genes pufB and pufA), of the reaction centre (genes pufL and pufM), a scaffold protein (gene pufX) and includes the gene for sRNA PcrX. Segmental differences in the stability of the pufBALMX-pcrX mRNA contribute to the stoichiometry of LHI to RC complexes. With asPcrL we identified the third sRNA and the first antisense RNA that is involved in balancing photosynthesis gene expression in R. sphaeroides. asPcrL influences the stability of the pufBALMX-pcrX mRNA but not of the pufBA mRNA and consequently the stoichiometry of photosynthetic complexes. By base pairing to the pufL region asPcrL promotes RNase III-dependent degradation of the pufBALMX-prcX mRNA. Since asPcrL is activated by the same protein regulators as the puf operon including PcrX it is part of an incoherent feed-forward loop that fine-tunes photosynthesis gene expression.[Figure: see text].

SUBMITTER: Reuscher CM

PROVIDER: S-EPMC8489922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3'-to-5' exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5'-to-3' end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3' end.ResultsThe PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3' ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3' end analysis revealed that 885 3' ends are degraded by PNPase. A fair percentage of these RNA 3' ends was also identified at the same genomic position in RNase E or RNase III mutant strains.ConclusionThe PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3' end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3' ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5'/3' ends. It is publicly available on GitHub and is distributed under ICS license.

Project description:Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the aa3 cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.

Project description:The Q gene of the facultative photoheterotroph Rhodobacter sphaeroides, localized immediately upstream of the oxygen- and light-regulated puf operon, encodes a 77-amino-acid polypeptide. The 5' and 3' ends of the 561-bp Q transcript were determined. To gain insight into the role of the Q gene product, a number of Q mutations were constructed by oligonucleotide-directed mutagenesis and subsequent substitution of the mutated form of the gene in single copy for the chromosomal copy via homologous recombination. The resulting mutants can grow photosynthetically, with the exception of QSTART, in which the initiation codon for the Q protein was altered. Spectral analysis of the intracytoplasmic membranes showed that one of the missense mutants (QdA) was deficient in the formation of detectable B875 light-harvesting complex (LHC), whereas deletion of the stem-loop structure (Qloop) failed to form B800-850 LHC when grown anaerobically either in the dark or under light intensity of 100 W/m2. Other missense mutants (QuA and QuB) contained either more B800-850 LHC or more B875 LHC, respectively, than the wild type. Although the levels of puf and puc transcripts isolated from QSTART grown anaerobically on succinate-dimethyl sulfoxide in the dark were comparable to wild-type levels, no B875 spectral complex was detected and there was a greater than 90% reduction in the level of the B800-850 pigment-protein complex. It has also been confirmed that the ultimate cellular levels of either the B875 or B800-850 spectral complexes can vary over wide limits without any change in the level(s) of complex specific transcripts. When the wild-type Q gene was reintroduced in trans into the Q mutations, QSTART was able to grow photosynthetically and both B800-850 and B875 spectral complexes were formed in either QdA or Qloop. Finally, we demonstrated that the level of each puf-specific mRNA behaves independently of one another as well as independently of the level(s) of Q gene-specific mRNA. These results are compatible with the existence of regulatory sequences affecting the puf mRNA level(s) being localized within the Q structural gene. These results suggest that Q-specific expression is uncoupled from puf-specific transcription and that the Q protein is not involved in the regulation of transcription of the puf operon but is directly involved in the assembly of both the B875 and B800-850 pigment-protein complexes.

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Antisense RNA asPcrL regulates expression of photosynthesis genes in Rhodobacter sphaeroides by promoting RNase III-dependent turn-over of puf mRNA.

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