Microbiome analysis of the restricted bacteria in radioactive element-containing water at the Fukushima Daiichi Nuclear Power Station.

A major incident occurred at the Fukushima Daiichi Nuclear Power Station following the tsunami triggered by the Tohoku-Pacific Ocean Earthquake in March 2011, whereby seawater entered the torus room in the basement of the reactor building. Here, we identify and analyze the bacterial communities in the torus room water and several environmental samples. Samples of the torus room water (1 × 109 Bq137Cs/L) were collected by the Tokyo Electric Power Company Holdings from two sampling points between 30 cm and 1 m from the bottom of the room (TW1) and the bottom layer (TW2). A structural analysis of the bacterial communities based on 16S rRNA amplicon sequencing revealed that the predominant bacterial genera in TW1 and TW2 were similar. TW1 primarily contained the genus Limnobacter, a thiosulfate-oxidizing bacterium. γ-Irradiation tests on Limnobacter thiooxidans, the most closely related phylogenetically found in TW1, indicated that its radiation resistance was similar to ordinary bacteria. TW2 predominantly contained the genus Brevirhabdus, a manganese-oxidizing bacterium. Although bacterial diversity in the torus room water was lower than seawater near Fukushima, ~70% of identified genera were associated with metal corrosion. Latent environment allocation-an analytical technique that estimates habitat distributions and co-detection analyses-revealed that the microbial communities in the torus room water originated from a distinct blend of natural marine microbial and artificial bacterial communities typical of biofilms, sludge, and wastewater. Understanding the specific bacteria linked to metal corrosion in damaged plants is important for advancing decommissioning efforts. IMPORTANCE: In the context of nuclear power station decommissioning, the proliferation of microorganisms within the reactor and piping systems constitutes a formidable challenge. Therefore, the identification of microbial communities in such environments is of paramount importance. In the aftermath of the Fukushima Daiichi Nuclear Power Station accident, microbial community analysis was conducted on environmental samples collected mainly outside the site. However, analyses using samples from on-site areas, including adjacent soil and seawater, were not performed. This study represents the first comprehensive analysis of microbial communities, utilizing meta 16S amplicon sequencing, with a focus on environmental samples collected from the radioactive element-containing water in the torus room, including the surrounding environments. Some of the identified microbial genera are shared with those previously identified in spent nuclear fuel pools in countries such as France and Brazil. Moreover, our discussion in this paper elucidates the correlation of many of these bacteria with metal corrosion.

Complete genome sequence of Limnobacter thiooxidans CS-K2T, isolated from freshwater lake sediments in Bavaria, Germany.

Limnobacter thiooxidans CS-K2T is a Gram-negative bacterium first isolated from the sediment of the littoral zone of a freshwater lake in Germany. We here present the complete annotated genome sequence of this thiosulfate-oxidizing bacterium, spanning 3.54 Mb and encoding 3,192 protein-coding sequences.

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance in Enterobacteriaceae.

Glutamine synthetase (GS) is the key enzyme of nitrogen assimilation induced under nitrogen limiting conditions. The carbon skeleton of glutamate and glutamine, 2-oxoglutarate, is supplied from the TCA cycle, but how this metabolic flow is controlled in response to nitrogen availability remains unknown. We show that the expression of the E1o component of 2-oxoglutarate dehydrogenase, SucA, is repressed under nitrogen limitation in Salmonella enterica and Escherichia coli. The repression is exerted at the post-transcriptional level by an Hfq-dependent sRNA GlnZ generated from the 3'UTR of the GS-encoding glnA mRNA. Enterobacterial GlnZ variants contain a conserved seed sequence and primarily regulate sucA through base-pairing far upstream of the translation initiation region. During growth on glutamine as the nitrogen source, the glnA 3'UTR deletion mutants expressed SucA at higher levels than the S. enterica and E. coli wild-type strains, respectively. In E. coli, the transcriptional regulator Nac also participates in the repression of sucA. Lastly, this study clarifies that the release of GlnZ from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA. Thus, the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Regulation of Transcription Termination of Small RNAs and by Small RNAs: Molecular Mechanisms and Biological Functions.

Accurate and efficient transcription termination is an important step for cells to generate functional RNA transcripts. In bacteria, two mechanisms are responsible for terminating transcription: intrinsic (Rho-independent) termination and Rho-dependent termination. Growing examples suggest that neither type of transcription termination is static, but instead are highly dynamic and regulated. Regulatory small RNAs (sRNAs) are key players in bacterial stress responses, are frequently expressed under specific growth conditions, and are predominantly terminated through the intrinsic termination mechanism. Once made, sRNAs can base-pair with mRNA targets and regulate mRNA translation and stability. Recent findings suggest that alterations in the efficiency of intrinsic termination for sRNAs under various growth conditions may affect the availability of a given sRNA and the ability of the sRNA to function properly. Moreover, alterations of mRNA structure, translation, and accessibility by sRNAs have the potential to impact the access of Rho factor to mRNAs and thus termination of the mRNA. Indeed, recent studies have revealed that some sRNAs can modulate target gene expression by stimulating or inhibiting Rho-dependent termination, thus expanding the regulatory power of bacterial sRNAs. Here we review the current knowledge on intrinsic termination of sRNAs and sRNA-mediated Rho-dependent termination of protein coding genes in bacteria.

Mechanism and physiological significance of autoregulation of the Escherichia coli hfq gene.

The RNA chaperone Hfq plays a critical role in sRNA-mediated gene regulation in enteric bacteria. The major role of Hfq is to stimulate base-pairing between sRNAs and target mRNAs by binding both RNAs through three RNA-binding surfaces. To understand the post-transcriptional network exerted by Hfq and its associated sRNAs, it is important to know how the cellular concentration of Hfq is regulated. While an early study showed that hfq translation is repressed by Hfq, the detailed mechanism and biological significance of the hfq autoregulation remain to be studied. Here, we show that the synthesis of Hfq is strictly autoregulated to maintain the cellular concentration of Hfq within a limited range even when the hfq mRNA is overexpressed from a plasmid-borne hfq gene. Mutational and biochemical studies demonstrate that Hfq represses its own translation primarily by binding to the hfq mRNA through the distal face. The growth of cells harboring the hfq plasmid is markedly inhibited due to an increased Hfq level when the distal face of Hfq is mutated or the 5'-UTR of hfq is mutated. A mutation in the rim suppresses the growth inhibition caused by the distal face mutation, suggesting that the interaction of Hfq with undefined RNAs through the rim is responsible for the growth inhibition by the increased Hfq level. In addition, the data suggest that the hfq autoregulation operates not only in cells harboring a multicopy hfq gene but also in the wild-type cells.

The effect of visual cues on performance in the ultimatum game in individuals with autism spectrum disorder.

The Ultimatum Game (UG) allows for the assessment of altruistic behavior as well as the perception of fairness. We examined the effects of visual social cues (gaze of others), factors associated with autism, and trust on UG performance in typical adults (TAs) and individuals with autism spectrum disorder (ASD). We hypothesized that individuals with ASD would be less affected by visual social cues than TAs. We recruited 30 TAs and 30 individuals with ASD. Participants completed 30 trials of the UG, during which the visual background was altered to include either stylized eyespots, flowers, or a neutral background. Reaction times and money distributed in each condition were recorded. Reaction times did not vary among background conditions in either group, although individuals with ASD responded more slowly overall. TAs distributed less money in the neutral background and flowers conditions than in the eyespots condition, while no significant differences in the amount of money distributed were observed among background conditions for individuals with ASD, who also distributed more money overall than TAs. Such findings may be due to decreased susceptibility to social cues among individuals with ASD.

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs.

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.

Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq.

An RNA chaperone of Escherichia coli, called host factor required for phage Qß RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity.

Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products.

The genes encoding Hfq-dependent sRNAs possess a typical Rho-independent transcription terminator. Here, we have studied the molecular events occurring at Rho-independent terminators of sRNA genes, focusing on two well-characterized Hfq-binding sRNAs, SgrS and RyhB. We constructed several hybrid genes in which the DNA sequence corresponding to a strong Rho-independent terminator was placed just downstream from the Rho-independent terminators of sRNA genes. By using this system, we demonstrate that transcripts frequently read through the Rho-independent terminators of sgrS and ryhB in normally growing cells. We show that Hfq does not affect the transcriptional readthrough event itself. We also find that the readthrough products no longer bind to Hfq in vivo. We have developed a competition assay based on a biotin-streptavidin system to analyze the interaction of Hfq and a particular RNA molecule in vitro. By using this method, we verify that the 3'-extended form of SgrS does not bind to Hfq in vitro. Finally, we demonstrate that transcription termination is significantly enhanced under stress conditions where transcription initiation of sRNA genes on the chromosome is induced. We conclude that the production of sRNAs is regulated not only at the step of transcription initiation but also at the step of transcription termination. The mechanism by which transcription termination is enhanced under stress conditions remains to be understood.

Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin.

Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels; and (ii) photo-sensors such as sensory rhodopsin from microbes and visual pigments from animals. Anabaena sensory rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial sensory rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light.

Detection of sRNA-mRNA interactions by electrophoretic mobility shift assay.

Electrophoretic mobility shift assay is a simple, rapid, and sensitive technique to analyze the RNA-RNA interaction. A (32)P-labeled RNA is incubated with another unlabeled RNA and subjected to electrophoresis on a native polyacrylamide gel. If two RNA molecules base pair stably, the movement of the probe RNA through the gel is retarded resulting in a characteristic band corresponding to the RNA duplex. Here, we describe the methods to study the interaction of an Hfq-binding small RNA (sRNA) and its target mRNA. Although we focus on the interaction of SgrS and its target ptsG mRNA, the methods can be applied to the analysis of base pairing between any sRNAs and their targets.

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3' poly(U) tail.

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.

PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action.

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.

RNase E action at a distance: degradation of target mRNAs mediated by an Hfq-binding small RNA in bacteria.

A major class of bacterial small RNAs (sRNAs), along with RNA-binding protein Hfq and endoribonuclease RNase E, acts on target mRNAs through base-pairing, leading to translational repression and rapid degradation of the mRNAs. In this issue of Genes & Development, Prévost and colleagues (pp. 385-396) demonstrate by using the well-characterized sRNA RyhB that RNase E cleavage at sites distal from the pairing region triggers degradation of target mRNAs. The study has provided an important insight into the initial events of sRNA-induced degradation of target mRNAs.

Hfq binding at RhlB-recognition region of RNase E is crucial for the rapid degradation of target mRNAs mediated by sRNAs in Escherichia coli.

An RNA chaperon Hfq along with Hfq-binding sRNAs stably binds to RNase E in Escherichia coli. The role of the Hfq-RNase E interaction is to recruit RNase E to target mRNAs of sRNAs resulting in the rapid degradation of the mRNA-sRNA hybrid. The C-terminal scaffold region of RNase E is responsible for the interaction with Hfq. Here, we demonstrate that the scaffold region can be deleted up to residue 750 without losing the ability to cause the rapid degradation of target mRNAs mediated by Hfq/sRNAs. The truncated RNase E750 can still bind to Hfq although the truncation significantly reduces the Hfq-binding ability. We conclude that the subregion between 711 and 750 is sufficient for the functional interaction with Hfq to support the rapid degradation of ptsG mRNA although additional subregions within the scaffold are also involved in Hfq binding. Deletion of the 702-750 region greatly impairs the ability of RNase E to cause the degradation of ptsG mRNA. In addition, a polypeptide corresponding to the scaffold region binds to Hfq without the help of RNA. Finally, we demonstrate that overexpression of RhlB partially inhibits the Hfq binding to RNase E and the rapid degradation of ptsG mRNA.

A minimal base-pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA.

Escherichia coli SgrS is an Hfq-binding small RNA that is induced under glucose-phosphate stress to cause translational repression and RNase E-dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31-nt-long stretch in the 3' region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre-annealed with ptsG mRNA by a high-temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base-pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base-pairing region is sufficient to inhibit ptsG translation in the context of full-length SgrS in vivo. We conclude that SgrS 168-181 is a minimal base-pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high-temperature pre-treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.

The Plasmodium HU homolog, which binds the plastid DNA sequence-independent manner, is essential for the parasite's survival.

The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU's physiological function. PfHU, which is targeted exclusively to the parasite's plastid, bound its natural target--the plastid DNA--sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite's survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.

RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA.

SgrS is an Hfq-binding small RNA that is induced under glucose phosphate stress in Escherichia coli. It forms a specific ribo nucleo protein complex with Hfq and RNase E resulting in translational repression and rapid degradation of ptsG mRNA, encoding the glucose transporter. Here, we report translational silencing of ptsG mRNA in a defined in vitro system. We demonstrate that SgrS and Hfq are the minimum components for translational silencing to faithfully reproduce the reaction in cells. We show that ptsG-SgrS base pairing is sufficient to cause translational repression when the ptsG mRNA is forced to base pair with SgrS without the help of Hfq. The extent of translational repression correlates with the extent of duplex formation. We conclude that base pairing itself but not Hfq is directly responsible for translational silencing and the major role of Hfq in gene silencing is to stimulate the base pairing between SgrS and ptsG mRNA. This simple mechanism is in striking contrast to miRNA action in eukaryote in which the RNA is believed to act only as a guide of protein partners.

Cleavage of mRNAs and role of tmRNA system under amino acid starvation in Escherichia coli.

We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin-antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions.