Biology subjects

Storz, G.

Publications and source records attributed to Storz, G..

16 recordsLinked to original sources

bioRxiv2026

AltORF-encoded microprotein recruits primary-ORF-encoded dehydrogenase to cell poles upon heat shock

Alternative open reading frames (altORFs) initiating from in-frame or out-of-frame start sites within annotated protein-coding genes represent a largely unexplored source of protein-coding potential and remain poorly understood in terms of regulation, function, and impact on physiology. Here we investigate Escherichia coli GndA, encoded by an out-of-frame altORF within gnd, which encodes 6-phosphogluconate dehydrogenase. We observe that the GndA protein is induced by heat shock, consistent with the initial identification in a quantitative membrane proteomics screen, but is unstable, while the levels of the stable Gnd dehydrogenase are unchanged. Using translational fusion assays and in vivo RNA structure probing, we show that an internal complementary sequence upstream of gndA acts as a thermo-responsive secondary RNA structure controlling GndA translation. Mutagenesis of either gnd or gndA reduce heat shock survival of E. coli indicating both gene products are important for resistance. GndA fractionates with membranes consistent with in silico predictions that the protein forms an amphipathic -helix. In contrast, Gnd is predominantly cytoplasmic but shows GndA-dependent relocalization to cell poles upon heat shock. Together, our results demonstrate that an altORF-encoded microprotein is required for stress-dependent relocalization of its primary ORF partner, revealing a functional interplay between proteins encoded by overlapping ORFs in bacterial stress adaptation.

microbiology↗

bioRxiv2026

Distribution of prophage-encoded Pas sRNAs across pathogenic Escherichia coli

Numerous base pairing small RNAs (sRNAs), which are an integral part of regulatory networks in bacteria, are encoded on mobile genetic elements (MGEs) in pathogenic strains of Escherichia coli. These sRNAs help coordinate the expression of MGE-encoded virulence factors with core genome-encoded cellular pathways. To investigate the evolution of MGE-encoded sRNAs, we queried public databases to characterize the distribution of PasA, PasB, PasC, PasD1, and PasD2, five prophage-encoded sRNAs discovered in enteropathogenic E. coli. We find that while the Pas sRNAs are largely restricted to pathogenic lineages of Escherichia and Shigella, they exhibit diversity in sequence, genomic presence, and copy number across strains. Based on phylogenetic analysis, the Pas sRNAs originate from multiple ancestral lineages and associate with specific E. coli pathovars, consistent with horizontal acquisition followed by retention. Syntenic analysis suggests a phage origin for the Pas sRNAs, likely from Shiga-toxin encoding phages, but the sRNAs appear to have diverged substantially following their integration into bacterial chromosomes. Comparative and structural analyses further suggest that the PasA and PasC sRNAs share a common ancestor as is the case for PasD and STnc100, another prophage-encoded sRNA. These findings add to our understanding of how accessory genome-encoded sRNAs emerge and evolve.

evolutionary biology↗

bioRxiv2025

RNA-RNA Interactome Approaches Provide in vivo Evidence for a Critical Role of the Hfq Rim Face in sRNA-mRNA Pairing

Most bacterial small regulatory RNAs (sRNAs) modulate gene expression by forming complementary base pairs with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq has three RNA-binding faces (proximal, rim, and distal), facilitating simultaneous binding of sRNAs and target mRNAs. Here, we systematically examined the functional impact of point mutations in the RNA binding faces on in vivo pairing, using the RNA Interaction by Ligation and Sequencing (RIL-seq) approach. The distal and proximal Hfq binding face mutants retained substantial numbers of RNA-RNA interactions (significant chimera counts or S-chimeras). However, the rim face mutant R16A showed a near-complete loss of S-chimeras, although Hfq R16A retained partial RNA binding activity as well as partial regulatory activity. Intracellular RIL-seq (iRIL-seq), a method with fewer in vitro processing steps, led to more S-chimeras in R16A but there were still fewer than for wild type Hfq and somewhat different sets of prevalent RNA-RNA pairs. Our analysis provides insights into how the RNA-binding faces of Hfq contribute to pairing in vivo, document the key role for the rim face in stabilizing RNA pairs on Hfq, and highlight intriguing differences captured by different RNA-RNA interactome approaches. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/660752v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@99569aorg.highwire.dtl.DTLVardef@104d130org.highwire.dtl.DTLVardef@141b117org.highwire.dtl.DTLVardef@1d19611_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase

Magnesium (Mg2+) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg2+ via conserved Mg2+ channels and transporters. The transporters are required for growth when Mg2+ is limiting or during bacterial pathogenesis, but, despite their significance, there are no known structures for these transporters. Here we report the first structure of the Mg2+ transporter MgtA solved by single particle cryo-electron microscopy (cryo-EM). Using mild membrane extraction, we obtained high resolution structures of both a homodimeric form (2.9 [A]), the first for a P-type ATPase, and a monomeric form (3.6 [A]). Each monomer unit of MgtA displays a structural architecture that is similar to other P-type ATPases with a transmembrane domain and two soluble domains. The dimer interface consists of contacts between residues in adjacent soluble nucleotide binding and phosphotransfer regions of the haloacid dehalogenase (HAD) domain. We suggest oligomerization is a conserved structural feature of the diverse family of P-type ATPase transporters. The ATP binding site and conformational dynamics upon nucleotide binding to MgtA were characterized using a combination of cryo-EM, molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and mutagenesis. Our structure also revealed a Mg2+ ion in the transmembrane segments, which, when combined with sequence conservation and mutagenesis studies, allowed us to propose a model for Mg2+ transport across the lipid bilayer. Finally, our work revealed the N-terminal domain structure and cytoplasmic Mg2+ binding sites, which have implications for related P-type ATPases defective in human disease.

biophysics↗

bioRxiv2024

Broadly conserved FlgV controls flagellar assembly and Borrelia burgdorferi dissemination in mice

Flagella propel pathogens through their environments yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly in the Lyme spirochete Borrelia burgdorferi, the archetypal flagellar regulator {sigma}28 is absent. We rediscovered gene bb0268 in B. burgdorferi as flgV, a broadly-conserved gene in the flagellar superoperon alongside {sigma}28 in many Spirochaetes, Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria. We found that B. burgdorferi FlgV is localized within flagellar motors. B. burgdorferi lacking flgV construct fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, B. burgdorferi lacking flgV survive and replicate in Ixodes ticks but are attenuated for dissemination and infection in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly.

microbiology↗

bioRxiv2023

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. SignificanceHere we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

microbiology↗

bioRxiv2023

Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen B. burgdorferi

Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. We employed complementary RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi - the etiological agent of Lyme disease. By systematically mapping B. burgdorferi RNA ends at single nucleotide resolution, we delineated complex gene arrangements and operons and mapped untranslated regions (UTRs) and small RNAs (sRNAs). We experimentally tested modes of B. burgdorferi transcription termination and compared our findings to observations in E. coli, P. aeruginosa, and B. subtilis. We discovered 63% of B. burgdorferi RNA 3' ends map upstream or internal to open reading frames (ORFs), suggesting novel mechanisms of regulation. Northern analysis confirmed the presence of stable 5' derived RNAs from mRNAs encoding gene products involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs resulted from premature termination and regulatory events, including forms of cis-acting regulation. For example, we documented that the polyamine spermidine globally influences the generation of truncated mRNAs. In one case, we showed that high spermidine concentrations increased levels of RNA fragments derived from an mRNA encoding a spermidine import system, with a concomitant decrease in levels of the full- length mRNA. Collectively, our findings revealed new insight into transcription termination and uncovered an abundance of potential RNA regulators.

microbiology↗

bioRxiv2022

A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3 untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3 UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblot and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ promotes survival of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

microbiology↗

bioRxiv2021

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

microbiology↗

bioRxiv2021

σ28-dependent small RNAs regulate timing of flagella biosynthesis

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor {sigma}28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3 UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

microbiology↗

bioRxiv2021

Synthetic dual-function RNA reveals features necessary for target regulation

Small base pairing RNAs (sRNAs) and small proteins comprise two classes of regulators that allow bacterial cells to adapt to a wide variety of growth conditions. A limited number of transcripts encoding both of these activities, regulation of mRNA expression by base pairing and a small regulatory protein, have been identified. Given that few have been characterized, little is known about the interplay between the two regulatory functions. To investigate the competition between the two activities, we constructed synthetic dual-function RNAs, hereafter referred to as MgtSR or MgtRS, comprised of the Escherichia coli sRNA MgrR and the open reading frame encoding the small protein MgtS. MgrR is a 98 nt base pairing sRNA that negatively regulates eptB encoding phosphoethanolamine transferase. MgtS is a 31 aa small inner membrane protein that is required for the accumulation of MgtA, a magnesium (Mg2+) importer. Expression of the separate genes encoding MgrR and MgtS is normally induced in response to low Mg2+ by the PhoQP two-component system. By generating various versions of this synthetic dual-function RNA, we probed how the organization of components and the distance between the coding and base pairing sequences contribute to the proper function of both activities of a dual-function RNA. By understanding the features of natural and synthetic dual-function RNAs, future synthetic molecules can be constructed to maximize their regulatory impact. IMPORTANCEDual-function RNAs encode a small protein and also base pair with mRNAs to act as small, regulatory RNAs. Given that only a limited number of dual-function RNAs have been characterized, further study of these regulators is needed to increase understanding of their features. This study demonstrates that a functional synthetic dual-regulator can be constructed from separate components and used to study the functional organization of dual-function RNAs, with the goal of exploiting these regulators.

microbiology↗

bioRxiv2021

AzuCR RNA modulates carbon metabolism as a dual-function RNA in Escherichia coli

Bacteria have evolved small RNAs (sRNAs) to regulate numerous biological processes and stress responses. While sRNAs generally are considered to be "noncoding", a few have been found to also encode a small protein. Here we describe one such dual-function RNA that modulates carbon utilization in Escherichia coli. The 164 nucleotide RNA was previously shown to encode a 28 amino acid protein (denoted AzuC). We discovered the membrane-associated AzuC protein interacts with GlpD, the aerobic glycerol-3-phosphate dehydrogenase, leading to increased GlpD activity. Overexpression of the RNA encoding AzuC results in a growth defect in glycerol and galactose medium. The defect in galactose medium was still observed for a stop codon mutant derivative, suggesting a potential regulatory role for the RNA. Consistent with this observation, we found that cadA and galE are repressed by base pairing with the RNA (denoted AzuCR). Interestingly, translation of AzuC interferes with the observed repression of cadA and galE by AzuCR and base pairing interferes with AzuC translation, demonstrating that the translation and base pairing functions compete.

microbiology↗

bioRxiv2020

Regulatory roles of 5' UTR and ORF-internal RNAs detected by 3' end mapping

Many bacterial genes are regulated by RNA elements in their 5' untranslated regions (UTRs). However, the full complement of these elements is not known even in the model bacterium Escherichia coli. Using complementary RNA-sequencing approaches, we detected large numbers of 3' ends in 5' UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We documented regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5' UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed that they have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5' UTRs and internal to ORFs.

microbiology↗

bioRxiv2019

Interactions of a bacterial RND transporter with a transmembrane small protein in a lipid environment

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases the resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that the drug sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate the activity of AcrB. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

microbiology↗

bioRxiv2019

Identifying small proteins by ribosome profiling with stalled initiation complexes

Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the true prevalence of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly-initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. The corresponding genes are not only intergenic, but are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.\n\nIMPORTANCEProteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the function of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

microbiology↗

bioRxiv2018

Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3' UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

microbiology↗