Phosphor imaging with the Sapphire helps identify a keystone sRNA in Pseudomonas aeruginosa

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In a recent study reported in PNAS, researchers from Harvard Medical School employed the phosphor imaging capabilities of the Azure Sapphire Biomolecular imager to image a Northern blot and the quantification from a Western blot from an Azure 600 Imager to identify potential RNA targets for the many small RNAs found in the opportunistic bacteria Pseudomonas aeruginosa (P. aeruginosa)1.

Since the release of this publication, the first generation Sapphire used has been succeeded by the new Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this new imager.

Small RNAs and RNA chaperones

Small RNAs (sRNA; also known as small regulatory RNAs) are important gene expression regulators in bacteria that have the ability to respond to environmental conditions. They do not code for proteins, but serve as regulatory molecules involved in fine-tuning gene activity through binding to target mRNAs. sRNA also either inhibit translation or affect mRNA stability. They play critical roles in diverse cellular processes, including development, stress response, and immune defense 2.

RNA chaperones are specialized proteins that facilitate proper RNA folding and assist in the binding of these sRNAs and their RNA targets. They act as molecular escorts, ensuring proper structure and function of RNA molecules in various cellular contexts 3. Together, sRNAs and RNA chaperones work to precisely regulate gene expression.

Pseudomonas aeruginosa as an Opportunistic Pathogen

P. aeruginosa is a prokaryotic gram-negative, opportunistic pathogen known for its ability to thrive and adapt across diverse environments. It is responsible for over 30,000 infections among hospital patients each year and is prone to developing antibiotic resistance4. Therefore, more research on P. aeruginosa is needed to improve treatment options. The survival and adaptability of P. aeruginosa are believed to rely on changes in the expression levels of regulatory proteins, including sRNAs, to the cellular environment.

 sRNAs regulate gene expression changes at the post-transcriptional level by binding to target mRNAs and influencing their ability to be translated into protein.

P. aeruginosa harbors well over one hundred putative sRNAs. However, the precise regulatory targets of most of these sRNAs remain largely unknown. Gebhardt et al. set out to identify some of these sRNA targets and to begin to decipher the network of regulatory interactions involving sRNAs in P. aeruginosa.

Hfq: A Global Post-Transcriptional Regulator in P. aeruginosa

To begin, the researchers looked at which sRNAs bound to the prominent post-transcriptional regulator Host factor for RNA phage Qβ replication, or Hfq. Hfq is a well studied RNA chaperone that plays an important regulatory role in many different bacteria, including P. aeruginosa. Hfq facilitates the crucial base pairing interactions between sRNAs and their corresponding mRNA targets by stabilizing sRNAs and modulating their capacity to engage with target transcripts. As Hfq mediates pairing between an sRNA and a target mRNA, it can affect the translation and stability dynamics of the target mRNA species.

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RIL-seq: Identifying sRNA-Target Interactions in P. aeruginosa

To gain insights into the interactions between sRNAs and their targets, researchers used RNA interaction through ligation and sequencing (RIL-seq) to identify targets of Hfq-associated sRNAs. After isolating Hfq-bound RNA, RIL-seq uses computational technology to map the captured sRNA-target RNA interactions. Using this approach, interaction partners for 89 sRNAs were identified. An abundance of these RNA-RNA interactions involved one particular sRNA named PhrS.

Exploring the role of PhrS in gene regulation in P. aeruginosa

Previously, PhrS was mainly thought to function through pairing with a single target mRNA to regulate the abundance of the MvfR protein. This transcriptional regulator is required for synthesizing signals involved in quorum sensing (i.e. cell-cell communication about bacterial population densities5). Gebhardt and colleagues found PhrS binds many sRNAs, and also explored the possibility that PhrS has a more complex regulatory role that originally thought.

While MvfR affects quorum sensing in P. aeruginosa, it is not the only transcriptional regulator to do so. The transcription activator AntR negatively regulates quorum sensing by activating expression of the antABC genes.  The researchers observed that expression of two of these antABC genes  was decreased when PhrS was ectopically expressed. Since ectopic expression of an sRNA can affect the ability of native sRNAs to compete for the same targets, the researchers investigated how native levels of PhrS influence expression of the antR, antA and antB genes.

Using qRT-PCR, expression of antR, antA, and antB was measured in both wild-type and ΔphrS mutant cells . Interestingly, the results revealed that in the absence of PhrS, the expression of these genes was significantly higher, ranging from 5 to 40 times higher compared to the wild-type cells. These findings indicate that PhrS negatively regulates these genes. Whether this negative regulation depends on direct interaction between PhrS and its antABC targets remained unknown.

To begin to address whether PhrS regulates the abundance of antR mRNA through direct interaction, the team created PhrS mutants with nucleotide substitutions in the region predicted to pair with antR mRNA (the “seed” region). These substitutions were intended to disrupt normal antR-PhrSA binding.

This experiment required the use of reporters to monitor PhrS-antR mRNA interactions. Through quantification of reporter activity, the team could assess whether PhrS and antR mRNA interact directly. Expression of the antR reporter increased in ΔphrS mutant cells compared to wild-type. Remarkably, the PhrS mutants failed to repress the reporter gene, while wild-type PhrS was able to do so.  This led the researchers to conclude that PhrS represesses the translation and/or stability of the antR mRNA and that the seed region is involved in this mechanism.

Since this paper was published, the Sapphire has been succeed by the new Sapphire FL

Designed for flexible choice in detection chemistry and samples, the Sapphire FL brings precise quantitation of nucleic acids and proteins
Scientist changing optical modules on the new Azure Sapphire FL
The new Azure Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

The usage of Northern and Western blots by Gebhardt et al

While the changes in antR expression seen in the ΔphrS mutant cells do indicate regulation of the gene by PhrS, they do not definitively demonstrate direct binding of PhrS to antR mRNA. To explore this further, the researchers created 10 different phrS mutants each with dinucleotide substitutions in the predicted seed region. They then used the same reporter assay as above to examine how each mutant phrS affected antR expression. Interestingly, they discovered that mutants with mutations between positions 178 and 181 had a reduced ability to negatively regulate antR reporter expression, with the mutation of position 179 to 180 (mutant SM179) having the most profound effect.

To ensure these results were not due to varying expression of these phrS mutants, a Northern blot detected using phosphor imaging by the Sapphire was imaged (Figure 4F). Northern blots are often performed to analyze gene expression of one or a small number of genes.

Northern blotting is a technique used by molecular biologists to detect a specific RNA sequence in a sample.

Northern blotting steps >

An advantage of using this type of botting analysis over other RNA analysis techniques, is that Northern blotting visualizes the size of RNA molecules which can reveal differences in processing, such as splicing variants. The probes used on Northern blots are small RNA or DNA nucleotides. Traditionally, probes are labeled with radioisotopes, which allows for quantitation of RNA species so that expression levels can be compared between samples.

The research team observed that all but one of the PhrS mutants were as abundant as the wildtype PhrS. These data support the conclusion that the PhrS seed sequence is required for direct regulation of the antR transcript.

Northern blot used to evaluate the levels of PhrS mutants captured using Azure Sapphire Imager from Gebhardt
Figure 4F from Gehardt et al. (2023). Representative Northern blot used to evaluate the levels of each PhrS mutant. The Azure Sapphire Biomolecular Imager was used to capture images of the Northern blot shown. Licensed under CC BY 4.0.

To this point, only antR reporters had been used to investigate the PhrS-antR relationship. The researchers sought to examine whether PhrS directly pairs with antR mRNA when the gene is in its native state position in the genome. To address this question, the researchers created a mutant cell line that snythesizes AntR from its native position, but includes a VSV-G epitope tag (AntR-V) for ease of detection. They also introduced a small change (called the M2C mutation) in a specific region of the antR gene. This mutation was designed to prevent the natural PhrS from binding to the gene, but it contained the complementary bases for a specific PhrS mutant called SM179.  Therefore, this M2C mutation would allow PhrS mutant SM179 to bind to (and presumably regulate) antR.  Additionally, they created another derivative cell line that could naturally produce the PhrS mutant SM179.

Western blots imaged using the Azure 600 (Figure 4G) revealed PhrS mutant SM179 reduced the abundance of the M2C version of AntR-V, validating the direct interaction of these mutants and the regulatory nature of the interaction.

Figure 4G from Gehardt et al. (2023). Representative Western blot showing the protein levels of AntR-V with and without the restorative mutant M2C and the PhrS- SM179 mutant. The Azure 600 Biomolecular Imager was used to capture images of the Western blot. Licensed under CC BY 4.0.

How PhrS affects the abundance of quorum sensing proteins through antR mRNA regulation

Since quorum sensing signals, such as PQS, are regulated by AntR, these findings suggest that PhrS may regulate quorum sensing through direct interaction with the antR transcript.  Indeed, the researchers found that, in absence of PhrS or in the presence of M2C antR mutants (where PhrS cannot bind to antR), expression of the quorum sensing molecule PQS was reduced . These findings suggest that PhrS is involved in quorum sensing through regulation of the transcriptional regulators responsible for quorum sensing.

The Ultimate Western Blot Imaging System

The Azure 600 offers laser technology with two IR detection channels enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.
Scientist choosing settings on Azure 600
The Azure 600 is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

Gebhardt et al discovered an extensive network of RNA-RNA interactions in P. aeruginosa involving Hfq, 89 unique sRNAs, and their target transcripts. PhrS was found to be involved in the regulation of hundreds of different transcripts and affects quorum-sensing molecules through a complex mechanism that was previously unknown.

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