Biology subjects

Wigneshweraraj, R.

Publications and source records attributed to Wigneshweraraj, R..

9 recordsLinked to original sources

bioRxiv2025

Condensation of the RNA chaperone Hfq is coupled to inhibition of carbon assimilation and contributes to the stabilisation of regulatory RNAs in nitrogen starved Escherichia coli

Ribonucleoprotein-condensates are membraneless compartments that concentrate RNA-binding proteins and RNA and play key roles in cellular adaptation across both eukaryotes and bacteria. While the biological roles of ribonucleoprotein-condensates are better understood in eukaryotic systems, the knowledge of metabolic processes that govern their formation and their contribution to stress adaptation remains at a nascent stage in bacterial RNA biology. Hfq is an RNA-chaperone conserved in many bacteria that undergoes condensation in response to diverse stresses. Using nitrogen (N) starvation in Escherichia coli as a model stress condition, we show that Hfq condensation occurs independently of any extracellular cues, cytoplasmic shrinkage that cells undergo during N starvation or the canonical NtrBC-dependent adaptive response to N starvation. However, we demonstrate that Hfq condensation is coupled to the inhibition of carbon assimilation in N-starved E. coli. Further, by comparing the transcriptomes of wild-type bacteria and bacteria unable to form Hfq-condensates, we reveal that Hfq-condensates contribute to the stabilisation of Hfq-associated non-coding regulatory RNAs. We propose that coordination of carbon and N metabolism during N starvation, critical for metabolic adaptation, is accompanied by preservation of non-coding regulatory RNAs via Hfq condensation.

microbiology↗

bioRxiv2024

Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

The RNA-chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal molecular architecture of the Hfq hexamer contains three well characterised surfaces which allow it to bind sRNAs to stabilise them and engage target transcripts. Hfq-interacting sRNAs are categorised into two classes based on the surfaces they use to bind Hfq. By characterising a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen starvation, we corroborated the important role of the three RNA binding surfaces for Hfq function. Surprisingly, we uncovered two conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfqs RNA binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilisation of both sRNA classes. However, the alanine substitutions at these residues had no measurable impact on protein stability, structure or equilibrium binding to target sRNAs in vitro. We propose that V22 and G34 are key to the cooperative function among the RNA-binding surfaces of Hfq, a mechanism especially critical under cellular conditions when there is an increased demand for Hfq.

microbiology↗

bioRxiv2023

Global RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery

The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA-RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.

microbiology↗

bioRxiv2023

Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more

Probiotic bacteria confer multiple health benefits, including preventing the growth, colonisation, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce McC from the wild-type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon, whilst ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little to none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic or diagnostic applications. IMPORTRANCEBacteria that resist killing by antibiotics are a major risk to modern medicine. The use of beneficial probiotic bacteria as chassis to make antibiotic-like compounds at the site of infection in the body is emerging as a popular alternative to the use of conventional antibiotics. A potential drawback of engineering probiotic bacteria in this way is that producing antibiotic-like compounds could impart undesired side-effects on the performance of such bacteria and thereby compromise their intended use. This study highlights considerations researchers may take into account when engineering probiotic bacteria for therapeutic, prophylactic or diagnostic applications.

microbiology↗

bioRxiv2022

The RNA polymerase gene specificity factor sigma 54 is required for homogeneous non-planktonic growth of uropathogenic Escherichia coli

The canonical function of a bacterial sigma ({sigma}) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the {sigma}54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the {sigma}54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. The genes that rely upon {sigma}54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by {sigma}54, is not yet exhaustive. By comparing the non-planktonic growth properties of prototypical enteropathogenic, uropathogenic and non-pathogenic Escherichia coli strains devoid of {sigma}54, we uncovered {sigma}54 as a determinant of homogenous non-planktonic growth specifically in the uropathogenic strain. Notably, bacteria devoid of individual activator ATPases of the {sigma}54-RNAP do not phenocopy the {sigma}54 mutant strain. It seems that {sigma}54s role as a determinant of homogenous non-planktonic growth represents a putative non-canonical function of {sigma}54 in regulating genetic information flow.

microbiology↗

bioRxiv2020

The adaptive response to long term nitrogen starvation in Escherichia coli requires the breakdown of allantoin

Bacteria initially respond to nutrient starvation by eliciting large-scale transcriptional changes. The accompanying changes in gene expression and metabolism allow the bacterial cells to effectively adapt to the nutrient starved state. How the transcriptome subsequently changes as nutrient starvation ensues is not well understood. We used nitrogen (N) starvation as a model nutrient starvation condition to study the transcriptional changes in Escherichia coli experiencing long-term N starvation. The results reveal that the transcriptome of N starved E. coli undergoes changes that are required to maximise chances of viability and to effectively recover growth when N starvation conditions become alleviated. We further reveal that, over time, N starved E. coli cells rely on the degradation of allantoin for optimal growth recovery when N becomes replenished. This study provides insights into the temporally coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. IMPORTANCEBacteria in their natural environments seldom encounter conditions that support continuous growth. Hence, many bacteria spend the majority of their time in states of little or no growth due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, bacteria have evolved several strategies, primarily manifesting themselves through changes in how the information in their genes is accessed. How these coping strategies change over time under nutrient starvation is not well understood and this knowledge is not only important to broaden our understanding of bacterial cell function, but also to potentially find ways to manage harmful bacteria. This study provides insights into how nitrogen starved Escherichia coli bacteria rely on different genes during long term nitrogen starvation.

microbiology↗

bioRxiv2020

The formation and function of focus-like structures of Hfq in long-term nitrogen starved Escherichia coli

The initial adaptive responses to nutrient depletion in bacteria often occur at the level of RNA metabolism. Hfq is an RNA-binding protein present in diverse bacterial lineages and contributes to many different aspects of RNA metabolism. We demonstrate that Hfq forms a distinct and reversible focus-like structure in E. coli specifically experiencing long-term nitrogen (N) starvation. Using the ability of T7 phage to replicate in N starved bacteria as a biological probe of E. coli cell function during N starvation, we demonstrate that Hfq foci have a role in the adaptive response to long-term N starvation. We further show that Hfq foci formation does not depend on gene expression during N starvation and occurs independently of the N regulatory protein C (NtrC) activated initial adaptive response to N starvation. The results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation. Significance StatementBacteria have evolved complex strategies to cope with conditions of nitrogen (N) adversity. We now reveal a role for a widely studied RNA binding protein, Hfq, in the processes involved in how Escherichia coli copes with N starvation. We demonstrate that Hfq forms a distinct and reversible focus-like structure in long-term N starved E. coli. We provide evidence to suggest that the Hfq foci are important features required for adjusting E. coli cell function during N starvation for optimal adaptation to long-term N starvation. The results have broad implications for our understanding of bacterial adaptive processes in response to stress.

microbiology↗

bioRxiv2019

Xenogeneic regulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

SPO1 phage infection of Bacillus subtilis results in a comprehensive remodelling of processes leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins for the specific shut down of various host processes including transcription, DNA synthesis and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module we identified eight gene products which attenuated B. subtilis growth. Out of the eight gene products that attenuated bacterial growth, a 25 kDa protein, called Gp53, was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Results reveal that Gp53 functions like a phage encoded adaptor protein and thereby appears to alter the substrate specificity of the ClpCP protease to modulate the proteome of the infected cell to benefit efficient SPO1 phage progeny development. It seems that Gp53 represents a novel strategy used by phages to acquire their bacterial prey.\n\nSignificance statementViruses of bacteria (phages) represent the most abundant living entities on the planet, and many aspects of our fundamental knowledge of phage-bacteria relationships remain elusive. Many phages encode specialised small proteins, which modulate essential physiological processes in bacteria in order to convert the bacterial cell into a factory for phage progeny production - ultimately leading to the demise of the bacterial cell. We describe the identification of several antibacterial proteins produced by a prototypical phage that infects Bacillus subtilis and describe how one such protein subverts the protein control system of its host to benefit phage progeny development. The results have broad implications for our understanding of phage-bacteria relationships and the therapeutic application of phages and their gene products.

microbiology↗

bioRxiv2018

A T7 phage factor required for managing RpoS in Escherichia coli

T7 development in Escherichia coli requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), E{sigma}70, by two T7 proteins: Gp2 and Gp5.7. While the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit E{sigma}S, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of buildup of guanosine pentaphosphate ((p)ppGpp) during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.\n\nSignificance statementVirus that infect bacteria (phages) represent the most abundant living entities on the planet and many aspects of our fundamental knowledge of phage-bacteria relationships have been derived in the context of exponentially growing bacteria. In the case of the prototypical Escherichia coli phage T7, specific inhibition of the housekeeping form of the RNA polymerase (E{sigma}70) by a T7 protein, called Gp2, is essential for the development of viral progeny. We now reveal that T7 uses a second specific inhibitor that selectively inhibits the stationary phase RNAP (E{sigma}S), which enables T7 to develop well in exponentially growing and stationary phase bacteria. The results have broad implications for our understanding of phage-bacteria relationships and therapeutic application of phages.

microbiology↗