Biology subjects

Vanderpool, C. K.

Publications and source records attributed to Vanderpool, C. K..

At least 19 recordsLinked to original sources

bioRxiv2026

A family of RRM-1 RNA binding proteins enables cold adaptation and environmental resilience in Bacteroides

Bacteria use post-transcriptional regulatory mechanisms to rapidly adjust gene expression during environmental change. In the gut-associated genus Bacteroides, these mechanisms remain poorly defined as these organisms lack canonical RNA chaperones like Hfq and CsrA that coordinate post-transcriptional stress responses in many well-studied model bacteria. Most Bacteroides possess conserved RNA recognition motif-1 (RRM-1) domain-containing RNA-binding proteins (more common in eukaryotes than bacteria) that have been proposed to act as global RNA chaperones. Here, we show that these RNA binding proteins (RBPs) are central to cold stress adaptation. Simultaneous deletion of all rbp genes produces a cold-sensitive growth defect across multiple Bacteroides species, while single deletions do not, revealing conserved functional redundancy. RBP transcripts and proteins accumulate rapidly after temperature downshift, and loss of RBPs extensively reprograms the transcriptome. Cold sensitivity of Bacteroides rbp mutants is not caused by defects in ribosome assembly or rRNA maturation. Instead, we find that in Bacteroides thetaiotaomicron, RBPs act together with BT1884, the sole canonical cold shock protein possessed by this organism. The combined loss of RBPs and BT1884 produces a synthetic severe cold sensitivity phenotype, defining two functionally redundant cold stress systems belonging to unrelated protein families. Strains lacking RBPs show reduced survival under simultaneous cold and oxygen stress, the conditions Bacteroides cells are expected to encounter during host-to-host transmission. Together, these findings establish RRM-1 RBPs as non-canonical cold shock proteins that enable cold adaptation and environmental survival in Bacteroides and suggest how these organisms withstand the stresses of transmission between hosts. IMPORTANCEBacteroides species are among the most abundant and stable members of the human gut microbiome, and they are also among the most readily transmitted between people. Reaching a new host requires surviving conditions outside the gut, including cold and oxygen exposure, yet how these bacteria withstand such stress is not well understood. Most bacteria manage stress using a well-defined set of RNA-binding proteins, but Bacteroides lack these canonical factors. We show that Bacteroides instead rely on a different family of RNA-binding proteins, more typical of eukaryotes than bacteria, to survive cold stress, and that these proteins promote survival under the conditions encountered during transmission. This work identifies a molecular system that allows an abundant and ecologically successful gut bacterium to endure the environmental challenges of moving between hosts.

microbiology↗

bioRxiv2024

The Salmonella pathogenicity island 1-encoded small RNA InvR mediates post-transcriptional feedback control of the activator HilA in Salmonella

Salmonella Pathogenicity Island 1 (SPI1) encodes a type three secretion system (T3SS) essential for Salmonella invasion of intestinal epithelial cells. Many environmental and regulatory signals control SPI1 gene expression, but in most cases, the molecular mechanisms remain unclear. Many of these regulatory signals control SPI1 at a post-transcriptional level and we have identified a number of small RNAs (sRNAs) that control the SPI1 regulatory circuit. The transcriptional regulator HilA activates expression of the genes encoding the SPI1 T3SS structural and primary effector proteins. Transcription of hilA is controlled by the AraC-like proteins HilD, HilC, and RtsA. The hilA mRNA 5 untranslated region (UTR) is ~350-nuclotides in length and binds the RNA chaperone Hfq, suggesting it is a likely target for sRNA-mediated regulation. We used the rGRIL-seq (reverse global sRNA target identification by ligation and sequencing) method to identify sRNAs that bind to the hilA 5 UTR. The rGRIL-seq data, along with genetic analyses, demonstrate that the SPI1-encoded sRNA InvR base pairs at a site overlapping the hilA ribosome binding site. HilD and HilC activate both invR and hilA. InvR in turn negatively regulates the translation of the hilA mRNA. Thus, the SPI1-encoded sRNA InvR acts as a negative feedback regulator of SPI1 expression. Our results suggest that InvR acts to fine-tune SPI1 expression and prevent overactivation of hilA expression, highlighting the complexity of sRNA regulatory inputs controlling SPI1 and Salmonella virulence. IMPORTANCESalmonella Typhimurium infections pose a significant public health concern, leading to illnesses that range from mild gastroenteritis to severe systemic infection. Infection is initiated and requires a complex apparatus that the bacterium uses to invade the intestinal epithelium. Understanding how Salmonella regulates this system is essential for addressing these infections effectively. Here we show that the small RNA (sRNA) InvR imposes negative feedback regulation on expression of the invasion system. This work underscores the role of sRNAs in Salmonellas complex regulatory network, offering new insights into how these molecules contribute to bacterial adaptation and pathogenesis.

microbiology↗

bioRxiv2024

Plasmid transmission dynamics and evolution of partner quality in a natural population of Rhizobium leguminosarum

Many bacterial traits important to host-microbe symbiosis are determined by genes carried on extrachromosomal replicons such as plasmids, chromids, and integrative and conjugative elements. Multiple such replicons often coexist within a single cell and, due to horizontal mobility, have patterns of variation and evolutionary histories that are distinct from each other and from the bacterial chromosome. In nitrogen-fixing Rhizobium, genes carried on multiple plasmids make up almost 50% of the genome, are necessary for the formation of symbiosis, and underlie bacterial traits including host plant benefits. Thus the genomics and transmission of plasmids in Rhizobium underlie the ecology and evolution of this important model symbiont. Here we leverage a natural population of clover-associated Rhizobium in which partner quality has declined in response to long-term nitrogen fertilization. We use 62 novel, reference-quality genomes to characterize 257 replicons in the plasmidome and study their genomics and transmission patterns. We find that, of the four most frequent plasmid types, two (types II & III) have more stable size, larger core genomes, and track the chromosomal phylogeny (display more vertical transmission), while others (types I & IV - the symbiosis plasmid, or pSym) vary substantially in size, shared gene content, and have phylogenies consistent with frequent horizontal transmission. We also find differentiation in pSym subtypes driven by long-term nitrogen fertilization. Our results highlight the variation in plasmid transmission dynamics within a single symbiont and implicate plasmid horizontal transmission in the evolution of partner quality. IMPORTANCEUnderstanding how bacterial genes move through natural populations is critical for understanding how bacterial traits evolve. The nitrogen-fixing bacterium Rhizobium leguminosarum lives in symbiosis with plants and is a model for studying how gene transmission from one cell to another on mobile genetic elements called plasmids impacts the evolution of bacteria and plants. Here we characterize the genomes of a natural bacterial population, then use novel approaches to show that mechanisms of plasmid gene transmission varies across multiple plasmid types possessed by R. leguminosarum. We find that changes in plasmid genes are associated with the decline of symbiotic partner quality in strains isolated from environments undergoing long-term fertilization. Together, these results underscore the importance of plasmid evolution in shaping ecosystem processes like nitrogen cycling. Our study provides a framework for probing the plasmid dynamics within natural bacterial populations and how plasmid transmission affects genetic diversity and ecological interactions in bacteria.

genomics↗

bioRxiv2024

Determinants of raffinose family oligosaccharide use in Bacteroides species

Bacteroides species are successful colonizers of the human gut and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in Polysaccharide Utilization Loci (PULs). While recent work has uncovered the PULs required for use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by -1,6 bonds to a sucrose (glucose -1-{beta}-2 fructose) moiety. Previous work showed that an -galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in subset and show that representative strains of species containing a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide. ImportanceThe gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the gut microbiota since they remain undigested by the host. Here, we study how the model gut commensal, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.

microbiology↗

bioRxiv2024

Transcriptional and post-transcriptional mechanisms modulate cyclopropane fatty acid synthase through small RNAs in Escherichia coli

The small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The conditions and regulators that control RydC synthesis had not previously been identified. In this study, we demonstrate that RydC regulation of cfa is important for resistance to membrane-disrupting conditions. We identify a GntR-family transcription factor, YieP, that represses rydC transcription and show that YieP indirectly regulates cfa through RydC. YieP positively autoregulates its own transcription. We further identify additional sRNA regulatory inputs that contribute to control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link control of membrane biophysical properties to multiple environmental signals. ImportanceBacteria experience many environmental stresses that challenge their membrane integrity. To withstand these challenges, bacteria sense what stress is occurring and mount a response that protects membranes. Previous work documented the important roles of small RNA (sRNA) regulators in membrane stress responses. One sRNA, RydC, helps cells cope with membrane-disrupting stresses by promoting changes in the types of lipids incorporated into membranes. In this study, we identified a regulator, YieP, that controls when RydC is produced, and additional sRNA regulators that modulate YieP levels and RydC activity. These findings illuminate a complex regulatory network that helps bacteria sense and respond to membrane stress.

microbiology↗

bioRxiv2024

Multiple Small RNAs Modulate Rho-Dependent Termination in the Cyclopropane Fatty Acid Synthase mRNA 5' Untranslated Region

Bacteria use a multi-layered regulatory strategy to precisely and rapidly tune gene expression in response to environmental cues. Small RNAs (sRNAs) form an important layer of gene expression control and most act post-transcriptionally to control translation and stability of mRNAs. We have shown that at least five different sRNAs in Escherichia coli regulate the cyclopropane fatty acid synthase (cfa) mRNA. These sRNAs bind at different sites in the long 5 untranslated region (UTR) of cfa mRNA and previous work suggested that they modulate RNase E-dependent mRNA turnover. Recently, the cfa 5 UTR was identified as a site of Rho-dependent transcription termination, leading us to hypothesize that the sRNAs might also regulate cfa transcription elongation. In this study we find that a pyrimidine-rich region flanked by sRNA binding sites in the cfa 5 UTR is required for premature Rho-dependent termination. We discovered that both the activating sRNA RydC and repressing sRNA CpxQ regulate cfa primarily by modulating Rho-dependent termination of cfa transcription, with only a minor effect on RNase E-mediated turnover of cfa mRNA. A stem-loop structure in the cfa 5 UTR sequesters the pyrimidine-rich region required for Rho-dependent termination. CpxQ binding to the 5 portion of the stem increases Rho-dependent termination whereas RydC binding downstream of the stem decreases termination. These results reveal the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and demonstrate that regulation by sRNAs in long UTRs can involve modulation of transcription elongation. ImportanceBacteria respond to stress by rapidly regulating gene expression. Regulation can occur through control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs in Escherichia coli regulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria.

microbiology↗

bioRxiv2023

Mechanisms of regulation of cryptic prophage-encoded gene products in Escherichia coli

The dicBF operon of Qin cryptic prophage in Escherichia coli K12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of the dicBF operon have remained unclear. Under standard laboratory growth conditions, transcription from dicBp, the major promoter of the dicBF operon, is repressed by DicA. Here, we discovered that transcription of the dicBF operon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronic dicBF mRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression of dicBp can be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of the dicBF operon and filamentation is mediated by DicF and DicB. Spontaneous derepression of dicBp occurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of {lambda} phage with DicA and DicC performing functions similar to CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of the dicBF operon. Collectively, our results illustrate that toxic genes encoded on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations. ImportanceCryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examine the regulation of a cryptic prophage operon in Escherichia coli encoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.

microbiology↗

bioRxiv2022

Small RNAs activate Salmonella pathogenicity island 1 by modulating mRNA stability through the hilD mRNA 3' UTR

Salmonella enterica serovar Typhimurium is an enteric pathogen associated with food-borne disease. Salmonella invades the intestinal epithelium using a type three secretion system encoded in Salmonella pathogenicity Island 1 (SPI-1). SPI-1 genes are tightly regulated by a complex feed-forward loop to ensure proper spatial and temporal expression. Most regulatory input is integrated at HilD, through control of hilD mRNA translation or HilD protein activity. The hilD mRNA possesses a 310-nucleotide 3' untranslated region (UTR) that influences HilD and SPI-1 expression, and this regulation is dependent on Hfq and RNase E, cofactors known to mediate small RNA (sRNA) activities. Thus, we hypothesized that the hilD mRNA 3' UTR is a target for sRNAs. Here we show that the sRNAs, SdsR and Spot 42 regulate SPI-1 by targeting different regions of the hilD mRNA 3' UTR. Regulatory activities of these sRNAs depend on Hfq and RNase E, in agreement with previous roles found for both at the hilD 3' UTR. We show that SdsR and RNase E are responsible for the accumulation of variable fragments of the hilD mRNA 3' UTR. Collectively, this work suggests that these sRNAs targeting the hilD mRNA 3' UTR regulate hilD mRNA levels by interfering with RNase E-dependent mRNA degradation. Our work provides novel insights into mechanisms of sRNA regulation at bacterial mRNA 3' UTRs and adds to our knowledge of post-transcriptional regulation of the SPI-1 complex feed-forward loop. ImportanceSalmonella are prominent food-borne pathogens, infecting millions of people a year. To express virulence genes at the correct time and place in the host, Salmonella uses a complex regulatory network that senses environmental conditions. Known for their role in allowing quick responses to stress and virulence conditions, we investigate the role of small RNAs in facilitating precise expression of these genes. We provide evidence that the 3' untranslated region of the hilD mRNA, encoding a key virulence regulator, is a target for small RNAs and the ribonuclease RNase E. The small RNAs play a role in stabilizing hilD mRNA to allow proper expression of Salmonella virulence genes in the host.

microbiology↗

bioRxiv2021

The sRNA MicC downregulates hilD translation to control the SPI1 T3SS in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. This binding blocks translation but does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other mechanisms. MicC-mediated regulation plays a role during infection, as evidenced by an increase in Salmonella fitness in the intestine in the micC deletion mutant that is dependent on the SPI1 T3SS. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor. IMPORTANCEThe Salmonella SPI1 T3SS is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.

microbiology↗

bioRxiv2021

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron

Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes. ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

microbiology↗

bioRxiv2020

Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

Small RNAs (sRNAs) are important regulators of gene expression in bacteria, particularly during stress responses. Many genetically and biochemically well characterized sRNAs regulate gene expression post-transcriptionally, by affecting translation and degradation of the target mRNA after they bind to their targets through base pairing. However, how regulation at each of these levels quantitatively contributes to the overall efficacy of sRNA-mediated regulation is not well understood. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation. Unexpectedly, our data reveal that certain previously characterized sRNAs are able to regulate some targets co-transcriptionally, rather strictly post-transcriptionally, and suggest that sRNA-mediated regulation can occur early in the mRNAs lifetime, perhaps as soon as the sRNA binding site is transcribed. In addition, our data suggest several important kinetic steps that may determine the efficiency and differential regulation of multiple mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA is likely the rate-limiting step and may dictate the regulation hierarchy observed within an sRNA regulon.

biophysics↗

bioRxiv2020

Effects of individual base-pairs on in vivo target search and destruction kinetics of small RNA

Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kinetics in vivo are influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target, ptsG mRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in the in vivo rates of target association, kon, and dissociation, koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreased kon and increased koff, providing an in vivo demonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced kon by 24-31% and increased koff by 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.

molecular biology↗

bioRxiv2020

Stochastic Analysis Probes the Role of the Chaperone Hfq in E. coli Sugar Shock Response

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or "sugar shock" in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

biophysics↗

bioRxiv2019

Thiamine pyrophosphate riboswitches in Bacteroides species regulate transcription or translation of thiamine transport and biosynthesis genes

Thiamine (vitamin B1) and its phosphorylated precursors are necessary for decarboxylation reactions required in carbohydrate and branched chain amino acid metabolism. Due to its critical roles in central metabolism, thiamine is essential for human and animal hosts and their resident gut microbes. However, little is known about how thiamine availability shapes the composition of gut microbial communities and the physiology of individual species within those communities. Our previous work has implicated both thiamine biosynthesis and transport activities in the fitness of Bacteroides species. To better understand thiamine-dependent gene regulation in Bacteroides, we examined thiamine biosynthesis and transport genes in three representative species: Bacteroides thetaiotaomicron, Bacteroides uniformis, and Bacteroides vulgatus. All three species possess thiamine biosynthetic operons controlled by highly conserved cis-acting thiamine pyrophosphate (TPP) riboswitches. B. thetaiotaomicron and B. uniformis have additional TPP riboswitch-controlled operons encoding thiamine transport functions. Transcriptome analyses showed that each Bacteroides species had a distinct transcriptional response to exogenous thiamine. Analysis of transcript levels and translational fusions demonstrated that in B. thetaiotaomicron, the TPP riboswitch upstream of biosynthesis genes acts at the level of transcription, while TPP riboswitches upstream of transport operons work at the level of translation. In B. uniformis and B. vulgatus, TPP riboswitches work at the transcriptional level to control downstream operons. The varying responses to exogenous thiamine and use of varied regulatory mechanisms may play an important role in niche establishment by the Bacteroidetes in the complex and constantly shifting gut environment. ImportanceBacteroides species are important and abundant members of human gut microbiome communities. Their activities in the gut are influenced by constant changes in nutrient availability. In this study, we investigated the genetic basis of thiamine (Vitamin B1) uptake and biosynthesis in three representative Bacteroides species. We found species-specific differences in the response to exogenous thiamine, and distinct mechanisms for regulation of uptake and biosynthesis gene expression. Our work implies that gut Bacteroides have evolved distinct strategies for making or acquiring an essential nutrient. These mechanisms may play an important role in the success of Bacteroides in establishing a niche within complex gut microbiome communities.

microbiology↗

bioRxiv2019

Cryptic prophage-encoded small protein DicB protects Escherichia coli from phage infection by inhibiting inner membrane receptor proteins

Bacterial genomes harbor cryptic prophages that have lost genes required for induction, excision from host chromosomes, or production of phage progeny. Escherichia coli K12 strains contain a cryptic prophage Qin that encodes a small RNA, DicF, and small protein, DicB, that have been implicated in control of bacterial metabolism and cell division. Since DicB and DicF are encoded in the Qin immunity region, we tested whether these gene products could protect the E. coli host from bacteriophage infection. Transient expression of the dicBF operon yielded cells that were ~100-fold more resistant to infection by {lambda} phage than control cells, and the phenotype was DicB-dependent. DicB specifically inhibited infection by {lambda} and other phages that use ManYZ membrane proteins for cytoplasmic entry of phage DNA. In addition to blocking ManYZ-dependent phage infection, DicB also inhibited the canonical sugar transport activity of ManYZ. Previous studies demonstrated that DicB interacts with MinC, an FtsZ polymerization inhibitor, causing MinC localization to mid-cell and preventing Z ring formation and cell division. In strains producing mutant MinC proteins that do not interact with DicB, both DicB-dependent phenotypes involving ManYZ were lost. These results suggest that DicB is a pleiotropic regulator of bacterial physiology and cell division, and that these effects are mediated by a key molecular interaction with the cell division protein MinC.\n\nImportanceTemperate bacteriophages can integrate their genomes into the bacterial host chromosome and exist as prophages whose gene products play key roles in bacterial fitness and interactions with eukaryotic host organisms. Most bacterial chromosomes contain \"cryptic\" prophages that have lost genes required for production of phage progeny but retain genes of unknown function that may be important for regulating bacterial host physiology. This study provides such an example - where a cryptic prophage-encoded product can perform multiple roles in the bacterial host and influence processes including metabolism, cell division, and susceptibility to phage infection. Further functional characterization of cryptic prophage-encoded functions will shed new light on host-phage interactions and their cellular physiological implications.

microbiology↗

bioRxiv2018

sRNA-target Prediction Organizing Tool (SPOT) integrates computational and experimental data to facilitate functional characterization of bacterial small RNAs

Small RNAs (sRNAs) post-transcriptionally regulate mRNA targets, typically under conditions of environmental stress. Although hundreds of sRNAs have been discovered in diverse bacterial genomes, most sRNAs remain uncharacterized, even in model organisms. Identification of mRNA targets directly regulated by sRNAs is rate-limiting for sRNA functional characterization. To address this, we developed a computational pipeline that we named SPOT for sRNA-target Prediction Organizing Tool. SPOT incorporates existing computational tools to search for sRNA binding sites, allows filtering based on experimental data, and organizes the results into a standardized report. SPOT sensitivity (Correctly Predicted Targets/Total Known Targets) was equal to or exceeded any individual method when used on 12 characterized sRNAs. Using SPOT, we generated a set of target predictions for the sRNA RydC, which was previously shown to positively regulate cfa mRNA, encoding cyclopropane fatty acid synthase. SPOT identified cfa along with additional putative mRNA targets, which we then tested experimentally. Our results demonstrated that in addition to cfa mRNA, RydC also regulates trpE and pheA mRNAs, which encode aromatic amino acid biosynthesis enzymes. Our results suggest that SPOT can facilitate elucidation of sRNA target regulons to expand our understanding of the many regulatory roles played by bacterial sRNAs.\n\nIMPORTANCESmall RNAs (sRNAs) regulate gene expression in diverse bacteria by interacting with mRNAs to change their structure, stability or translation. Hundreds of sRNAs have been identified in bacteria, but characterization of their regulatory functions is limited by difficulty with sensitive and accurate identification of mRNA targets. Thus, new robust methods of bacterial sRNA target identification are in demand. Here, we describe our Small RNA-target Prediction Organizing Tool, which streamlines the process of sRNA target prediction by providing a single pipeline that combines available computational prediction tools with customizable results filtering based on experimental data. SPOT allows the user to rapidly produce a prioritized list of predicted sRNA-target mRNA interactions that serves as a basis for further experimental characterization. This tool will facilitate elucidation of sRNA regulons in bacteria, allowing new discoveries regarding the roles of sRNAs in bacterial stress responses and metabolic regulation.

microbiology↗

bioRxiv2018

Multiple Small RNAs Alter Membrane Lipid Composition via Post-Transcriptional Regulation of Cyclopropane Fatty Acid Synthase

Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that three additional sRNAs, ArrS, CpxQ, and GadF also regulate cfa post-transcriptionally. RydC, ArrS, and GadF act through masking an RNase E cleavage site in the cfa mRNA 5 untranslated region (UTR), and all three sRNAs post-transcriptionally activate cfa. In contrast, CpxQ binds to a different site in the cfa mRNA 5 UTR and represses cfa. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA.

microbiology↗

bioRxiv2017

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

The mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.\n\nIMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of \"one sRNA-one target\", and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

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