Protein Hfq Research Articles

Vibrio cholerae YaeO is a Structural Homologue of RNA Chaperone Hfq that Inhibits Rho-dependent Transcription Termination by Dissociating its Hexameric State

Rho-dependent transcription termination is a well-conserved process in bacteria. The Psu and YaeO proteins are the two established inhibitors of the ATP-dependent RNA helicase Rho protein of Escherichia coli. Here, we show a detailed sequence and phylogenetic analysis demonstrating that Vibrio cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO induces significant growth defect on in vivo expression and inhibits in vitro functions of the V. cholerae Rho on directly binding to the latter. Through various biophysical techniques, we showed that interaction of VcYaeO disrupts the oligomeric state of the VcRho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR structure of the EcYaeO. Interestingly, VcYaeO structurally resembles the Hfq protein, and like the latter, it exhibits ssDNA/RNA-binding properties. Docking studies demonstrate probable interactions of VcYaeO with VcRho and mode of inhibition of RNA binding to Rho. We propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter's hexameric assembly and also likely by sequestering the RNA from the Rho primarybinding sites.

Rewiring the functional complexity between Crc, Hfq and sRNAs to regulate carbon catabolite repression in Pseudomonas.

Pseudomonas species are the most versatile of all known bacteria for metabolic flexibility and the extent of host range from plants to humans that remains unmatched. The evolution of diverse metabolic strategies in these species to adapt to the fluctuating environment guarantees high fitness as well as the ability to withstand stress at multiple levels. These abilities in Pseudomonas species are imprinted by an adaptable genetic repertoire through the integration of external and internal signals via complex regulatory networks. One of the main regulatory networks that lead to optimal growth, survival and cellular robustness is the phenomenon of carbon catabolite repression (CCR). Even though a large array of information is available, the molecular machinery and the mechanism of CCR in Pseudomonas are distinctly diverse from Escherichia coli and Bacillus subtilis. In Pseudomonas, the Crc and Hfq proteins, CbrAB two-component systems and the CrcZ/CrcY small RNA are key components of CCR. The main focus of this review is to elucidate the mechanism of CCR and the accessories involved in regulation of preferred carbon source utilisation over non-preferred ones and how CCR influences the virulence, antibiotic resistance, bioremediation and plant growth promotion pathways. Furthermore, we have also tried to shed some light on the "omics" approaches which can provide deep mechanistic insights into the regulation of CCR. Understanding the mechanistic picture of key regulatory entities and mechanism responsible for metabolic flexibility will create opportunities for exploitation of these versatile prokaryotes in several biotechnological processes.

Regulation of metE+ mRNA expression by FnrS small RNA in Salmonella enterica serovar Typhimurium

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE+ gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS- and hfq- deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE+-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE+ mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5′ mRNA translational initiation region (TIR) of the metE+ gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE+ TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE+ 5′ TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE+ gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.

Hfq Globally Binds and Destabilizes sRNAs and mRNAs in Yersinia pestis.

Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3' regions of mRNAs. An Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq-binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5'P-activated RNase E degradation pathway, which is consistent with a model in which Hfq facilitates sRNA-mRNA base pairing and the coupled degradation in Y. pestis These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis IMPORTANCE Discovered in 1968 as an Escherichia coli host factor that was essential for replication of the bacteriophage Qβ, the Hfq protein is a ubiquitous and highly abundant RNA-binding protein in many bacteria. With the assistance of Hfq, small RNAs in bacteria play important roles in regulating the stability and translation of mRNAs by base pairing. In this study, we want to elucidate the Hfq-assisted sRNA-mRNA regulation in Yersinia pestis A global map of Hfq interaction sites in Y. pestis was obtained by sequencing cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could bind to hundreds of sRNAs and the majority of mRNAs in Y. pestis The enriched binding motifs in sRNAs and mRNAs are complementary to each other, suggesting a general base-pairing mechanism for sRNA-mRNA interaction. The Hfq-bound sRNA and mRNA regions were both destabilized. The results suggest that Hfq binding facilitates sRNA-mRNA base pairing and coordinates their degradation, which might enable Hfq to surveil the homeostasis of most mRNAs in bacteria.

New molecular interactions broaden the functions of the RNA chaperone Hfq.

The RNA chaperone Hfq is an important bacterial post-transcriptional regulator. Most studies on Hfq are focused on the role of this protein on small non-coding RNAs (sRNAs) and messenger RNAs (mRNAs). The most well-characterized function of Hfq is its role as RNA matchmaker, promoting the base-pairing between sRNAs and their mRNA targets. However, novel substrates and previous unrecognized functions of Hfq have now been identified, which expanded the regulatory spectrum of this protein. Hfq was recently found to bind rRNA and act as a new ribosome biogenesis factor, affecting rRNA processing, ribosome assembly, translational efficiency and translational fidelity. Hfq was also found to bind tRNAs, which could provide an additional mechanism for its role on the accuracy of protein synthesis. The list of substrates does not include RNA exclusively since Hfq was shown to bind DNA, playing an important role in DNA compaction. Additionally, Hfq is also capable to establish many protein-protein interactions. Overall, the functions of the RNA-binding protein Hfq have been expanded beyond its function in small RNA-mediated regulation. The identification of additional substrates and new functions provides alternative explanations for the importance of the chaperone Hfq as a global regulator.

Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.

RNA chaperone hfq mediates persistence to multiple antibiotics in Aeromonas veronii

Pathogenic Aeromonas veronii results in great healthy and economic losses in fishes and human. The multiple drug tolerance of bacterial persister is the major cause for recurrent infections. Ubiquitous RNA-binding protein Hfq is liable for antibiotic tolerance and persisiter production. We showed that the hfq deletion in A. veronii retarded the growth, reduced the tolerances to diverse antibiotics, and lowered the persistence. Such effects might be mediated by the downregulations of RelE, CspD, ClpB, RpoS, OxyR, and upregulation of OppB. Our study supports the role of Hfq in persister formation and provides clues for the avoidance of recalcitrant infections.

Effect of <italic>hfq2</italic> gene in <italic>Aeromonas hydrophila</italic> on biofilm formation

The RNA-binding protein Hfq is an evolutionarily conserved sRNA chaperone protein that can regulate the RNA translation at the post-transcription level and is involved in the regulation of important physiological functions in bacteria. The Aeromonas hydrophila ATCC 7966 genome contains two copies of the hfq gene ( AHA_0924 and AHA_3797 ). AHA_0924 has been found to share pronounced homology with many popular bacterial species and its biological functions was well documented. However, only few bacterial species carry the second hfq gene copy and its role remains largely unknown. To better understand the physiological function of AHA_3797 ( hfq2 ), we first successfully knocked out hfq2 using homologous recombination technology and found that its biofilm formation capability was significantly reduced in this study ( P ∆hfq2 in biofilm status. Our results showed a total of 1538 proteins, including 131 proteins that were up-regulated and 140 proteins that were down-regulated. Selected altered proteins were validated by Western blot analysis, which indicated the proteomics results were reliable. The following Gene Ontology (GO) annotation enrichment analysis in molecular functional clustering showed that up-regulated proteins were mainly involved in receptor activity, transaminase activity, and metal ion binding function, while down-regulated proteins were mainly involved in oxidoreductase activity. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the most plentiful up-regulated proteins tended to be involved in the biosynthesis of antibiotics, while the down-regulated proteins mainly involved chemotaxis of bacteria. We also found ∆hfq2 caused much of the up-regulation of bacterial iron transport-related proteins, such as colicin I receptor and TonB-dependent copper receptor, and down-regulation of chemotaxis-associated proteins, such as CheY, in the biofilm. The deletion of hfq2 did not result in a significant change in the expression of homologous Hfq protein, suggesting that the roles of both Hfq in the biofilm formation process of A. hydrophila are different. To verify the role of iron homeostasis during hfq2 -mediated biofilm formation, we compared the biofilm formation between ∆hfq2 and the wide-type strain under iron limitation conditions. The results showed that the biofilm formation of the control strain decreased, whereas that of ∆hfq2 i ncreased significantly after the addition of 200 μmol/L ferrous iron chelating 2,2′-dipyridyl (DIP), suggesting that Hfq2 protein negatively regulates intracellular iron homeostasis and affects biofilm formation. In general, this is the first work to confirm that the deletion of the hfq2 gene of A. hydrophila affects the formation of biofilm, suggesting that this gene is not a pseudogene, but rather it plays an important regulatory role in the formation of bacterial biofilm. Establishing the mechanism by which Hfq2 protein regulates bacterial biofilm formation would be of considerable scientific significance and may be helpful in the prevention and control of pathogenic bacteria and the development of new antibacterial strategies in the future.

A Research‐Driven Laboratory and Literature Course for Senior Biochemistry/Molecular Biology Undergraduates

Biochemistry/molecular biology (BMB) course‐based undergraduate research experiences (CUREs) are part of a growing movement to involve a greater number of undergraduates in authentic research experiences. Positive outcomes of CUREs are manifold, including improvement in scientific aptitude, confidence, and critical thinking skills that make students more likely to become career scientists. Here we describe the structure and outcomes of a one‐semester, senior BMB capstone CURE that pairs two 75‐min weekly discussion/problem‐solving sessions with semester‐long, weekly laboratory research. Within the context of two complex systems (bacterial and influenza infections), students were taught how to approach BMB literature. In the first three weeks of the course, students read and discussed primary literature directly relevant to their laboratory project. Additional literature selected for its demonstration of BMB techniques was presented by students throughout the remainder of the term in an alternating seminar/problem solving format. In the laboratory, all of the students studied homologs of the RNA‐binding protein Hfq, which is small (~11 kDa), thermostable, and present in about half of sequenced bacterial species. All students expressed and purified a previously uncharacterized, wild type Hfq homolog, enabling them to improve their technique with some instructor guidance. Simultaneously, students used databases and primary literature to design independent projects to test their own hypotheses about Hfq from various bacterial species. All pairs of students cloned novel proteins using synthetic biology and successfully purified Hfq at least once. This scaffolded approach to experimentation contextualized fundamental BMB technical skills, boosting understanding of sequence databases, cloning, SDS‐PAGE, protein expression/purification, three‐dimensional protein structure, and activity assays. Final laboratory papers were written using a manuscript submission template from a professional journal and a class‐wide, blinded peer review, furthering the emphasis on an authentic research experience. Students gained an appreciation for the medical impact of basic science and a multidimensional understanding of our discipline. This type of integrated course can be easily customized to emphasize other themes (e.g. cancer) and enhance undergraduate contributions to other original research projects within the time frame of a single semester.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Hfq-binding RNAs in Caulobacter crescentus

ABSTRACTSmall RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.

In Situ Characterization of Hfq Bacterial Amyloid: A Fourier-Transform Infrared Spectroscopy Study.

Hfq is a bacterial protein that regulates gene expression at the post-transcriptional level in Gram-negative bacteria. We have previously shown that Escherichia coli Hfq protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure in vitro. In the present work, we present evidence that Hfq unambiguously forms amyloid structures also in vivo. Taking into account the role of this protein in bacterial adaptation and virulence, our work opens possibilities to target Hfq amyloid self-assembly and cell location, with important potential to block bacterial adaptation and treat infections.

Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones.

Bacterial small regulatory RNAs (sRNAs) are the most abundant class of post-transcriptional regulators and have been well studied in Gram-negative bacteria. Little is known about the functions and mechanisms of sRNAs in high GC Gram-positive bacteria including Mycobacterium and Streptomyces. Here, we performed an in-depth study of 6C sRNA of Mycobacterium tuberculosis, which is conserved among high GC Gram-positive bacteria. Forty-seven genes were identified as possible direct targets of 6C sRNA and 15 of them were validated using an in vivo translational lacZ fusion system. We found that 6C sRNA plays a pleotropic role and regulates genes involved in various cellular processes, including DNA replication and protein secretion. Mapping the interactions of 6C sRNA with mRNA targets panD and dnaB revealed that the C-rich loops of 6C sRNA act as direct binding sites to mRNA targets. Unlike in Gram-negative bacteria where RNA binding proteins Hfq and ProQ are required, the interactions of 6C sRNA with mRNAs appear to be independent of RNA chaperones. Our findings suggest that the multiple G–C pairings between single stranded regions are sufficient to establish stable interactions between 6C sRNA and mRNA targets, providing a mechanism for sRNAs in high GC Gram-positive bacteria.

Function and Dynamics of the LSm2-8 Protein Ring during Spliceosome Activation

LSm2-8, a heteroheptameric protein ring, decorates the 3′ end of the U6 snRNA and is a stable component of U6 snRNP, U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and the spliceosome B complex. However, it is released during spliceosome activation, during which the active site for pre-mRNA splicing is created. While Lsm2-8 is known to stabilize the U6 snRNA in the cell, any potential function during spliceosome assembly or activation is unclear. Here, we characterize Lsm2-8 ejection during spliceosome activation using Colocalization Single Molecule Spectroscopy (CoSMoS). Our CoSMoS data reveals that LSm ring leaves after both release of U4 snRNP and arrival of Nine Teen Complex (NTC) on pre-mRNA. This indicates that a conformational change must occur within the NTC after spliceosome binding since available cryo-EM structures indicate that Lsm2-8 and NTC binding are mutually exclusive with one another. Thus, our data provides evidence for an intermediate spliceosome complex between B and Bact that has not been yet observed by cryo-EM. This unexpected late release of the Lsm2-8 ring also suggests an extended role during spliceosome activation. Based on sequence homology to bacterial Hfq proteins, it is possible that Lsm2-8 may be promoting formation of a RNA duplex between the U2 and U6 snRNAs. Using in vitro annealing assay, we observed that LSm ring forms a ternary complex in the presence U6 and U2 RNAs, which is dependent on base pairing. These results indicate that LSm ring may help in stabilizing U2/U6 helix II during activation and is retained on spliceosome until U4 unwinding is complete. After U4 is unwound from U6, LSm is replaced by NTC components that takeover stabilization of helix II during later stages of activation and splicing.

SWATH based quantitative proteomics analysis reveals Hfq2 play an important role on pleiotropic physiological functions in Aeromonas hydrophila

The RNA-binding protein Hfq protein is a well-characterized post-transcriptional regulator and plays an important role in the regulation of various physiological functions. Most bacterial genomes have only one copy of hfq, but a few bacterial species carry another distinct copy of hfq (hfq2) on the chromosome. However, the physiological properties of Hfq2 remain elusive. Here, we successfully constructed an hfq2 knock-out strain of Aeromonas hydrophila ATCC 7966. Phenotype assays showed that hfq2 deletion significantly increased extracellular protease activity, chemotaxis and swarming motility; increased low temperature, acidic pH, and basic pH resistance; and increased sensitivity to H2O2 stress and high temperatures. A SWATH-based quantitative proteomics method was used to compare the differential expression of proteins between the ∆hfq2 mutant and the wild-type strain. Bioinformatic analysis showed that proteins associated with metabolic pathways were mostly upregulated, while those associated with ribosome subunits were mostly downregulated. Moreover, the deletion of hfq2 leads to the increased expression of several DNA- or RNA-binding regulators, including Hfq and the catabolite gene activator (Crp), and the decreased expression of OmpR. To our knowledge, this is the first study to demonstrate the effects of Hfq2 on physiological function at the protein level. Biological significanceMost of bacterial genome has only one hfq copy, while only few bacterial species have two distinct copies in chromosome, and there are few documents about the biological functions of Hfq2. The current phenotype assays showed that Hfq2 plays important roles on bacterial physiological functions such as chemotaxis, swarming motility, ECPase activity and response to various environmental stresses. To better understanding the biological behavior of this protein, a SWATH based quantitative proteomics method was used to compare the altered proteins between ∆hfq2 and wide type strain. Bioinformatics analysis showed that ∆hfq2 significantly affects central metabolic pathway and translation related proteins. Moreover, the deletion of hfq2 lead to the increased expression of post-transcriptional regulator Hfq and catabolite gene activator Crp, and the down regulation of two-component regulatory system regulator OmpR. Our results indicate that Hfq2 is not a pseudogene but plays important roles on the essential physiological functions in A. hydrophila. To our knowledge, this is the first report to demonstrate the molecular function of Hfq2 at proteomics level.

A journey through the evolutionary diversification of archaeal Lsm and Hfq proteins.

Sm-like (Lsm) proteins are found in all three domains of life. They are crucially involved in the RNA metabolism of prokaryotic organisms. To exert their function, they assemble into hexa- or heptameric rings and bind RNA via a conserved binding pocket for uridine stretches in the inner pore of the ring. Despite the conserved secondary structure of Lsm proteins, there are several features that lead to a structural diversification of this protein family that mediates their participation in a variety of processes related to RNA metabolism. Until recently, the cellular function of archaeal Sm-like proteins was not well understood. In this review, we discuss structural features of Lsm proteins with a strong focus on archaeal variants, reflect on the evolutionary development of archaeal Lsm proteins and present recent insights into their biological function.

The Roles of Two hfq Genes in the Virulence and Stress Resistance of Burkholderia glumae.

The Hfq protein is a global small RNA chaperone that interacts with regulatory bacterial small RNAs (sRNA) and plays a role in the post-transcriptional regulation of gene expression. The roles of Hfq in the virulence and pathogenicity of several infectious bacteria have been reported. This study was conducted to elucidate the functions of two hfq genes in Burkholderia glumae, a causal agent of rice grain rot. Therefore, mutant strains of the rice-pathogenic B. glumae BGR1, targeting each of the two hfq genes, as well as the double defective mutant were constructed and tested for several phenotypic characteristics. Bacterial swarming motility, toxoflavin production, virulence in rice, siderophore production, sensitivity to H2O2, and lipase production assays were conducted to compare the mutant strains with the wild-type B. glumae BGR1 and complementation strains. The hfq1 gene showed more influence on bacterial motility and toxoflavin production than the hfq2 gene. Both genes were involved in the full virulence of B. glumae in rice plants. Other biochemical characteristics such as siderophore production and sensitivity to H2O2 induced oxidative stress were also found to be regulated by the hfq1 gene. However, lipase activity was shown to be unassociated with both tested genes. To the best of our knowledge, this is the first study to elucidate the functions of two hfq genes in B. glumae. Identification of virulence-related factors in B. glumae will facilitate the development of efficient control measures.

The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.

MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli. MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ, by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA. The mutation in the distal face slowed the ternary complex formation and affected the release of MgrR-mRNA complexes from Hfq, while the mutation in the proximal face weakened the MgrR binding to Hfq and in this way affected the annealing. Moreover, competition assays established that MgrR bound to both faces of Hfq and competed against other sRNAs. Further studies showed that uridine-rich sequences located in less structurally stable regions served as Hfq binding sites in each mRNA. Overall, the data show that the binding of MgrR sRNA to both faces of the Hfq ring enables it to efficiently anneal to target mRNAs. It also confers on MgrR a competitive advantage over other sRNAs, which could contribute to efficient cellular response to changes in magnesium homeostasis.

Signal transduction-dependent small regulatory RNA is involved in glutamate metabolism of the human pathogen Bordetella pertussis.

Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida

Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.

NMR resonance assignments for a ProQ homolog from Legionella pneumophila.

Regulation of gene expression on a post-transcriptional level by small non-coding regulatory RNAs (sRNAs) is very common in bacteria. sRNAs base pair with sequences in their target messenger RNAs (mRNAs) and thereby regulate translation initiation or mRNA stability. Specialized RNA-binding proteins (RBPs) facilitate these regulatory sRNA/mRNA interactions by acting as RNA chaperones. A well-known example for such an RNA chaperone which is widespread in bacteria is the Hfq protein. Recently, the ProQ/FinO protein family was identified as a new class of RNA chaperones involved in sRNA based regulation. Only a few members of this protein family have been structurally characterized so far. In particular, the structural basis for RNA-binding and recognition has not yet been established for this class of proteins. Here, we report the 1H, 13C and 15N NMR resonance assignments for a ProQ homolog (Lpp 1663) from the gram-negative pathogenic bacterium Legionella pneumophila which will facilitate further structural and dynamic studies of this protein and its interaction with RNA targets.