Genetic dissection of the signaling pathway required for the cell wall integrity checkpoint.

The cell wall integrity checkpoint monitors synthesis of cell wall materials during the Saccharomyces cerevisiae cell cycle. Upon perturbation of cell wall synthesis, the cell wall integrity checkpoint is activated, downregulating Clb2 transcription. Here, we identified genes involved in this checkpoint by genetic screening of deletion mutants. In addition to the previously identified dynactin complex, the Las17 complex, in particular the Bzz1 and Vrp1 components, plays a role in this checkpoint. We also revealed that the high osmolarity glycerol (HOG) and cell wall integrity mitogen-activated protein kinase (MAPK) signaling pathways are essential for checkpoint function. The defective checkpoint caused by the deficient dynactin and Las17 complexes was rescued by hyperactivation of the cell wall integrity MAPK pathway, but not by the activated form of Hog1, suggesting an order to these signaling pathways. Mutation of Fkh2, a transcription factor important for Clb2 expression, suppressed the checkpoint-defective phenotype of Las17, HOG MAPK and cell wall integrity MAPK mutations. These results provide genetic evidence that signaling from the cell surface regulates the downstream transcriptional machinery to activate the cell wall integrity checkpoint.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats.

DNA sequences that read the same from 5' to 3' in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as 'start codons'). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced N-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C4 to C18 in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C20 from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium Rhodovulum sulfidophilum. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C18-homoserine lactone (HSL), was also found. Among the detected C20-HSLs, 3-OH-C20-HSL was structurally identified and 3-OH-C20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C20 acyl side chain found to date. ABBREVIATIONS: AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography.

Magnesium chloride and polyamine can differentiate mouse embryonic stem cells into trophectoderm or endoderm.

Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations.

Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth.

The Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved.

Extracellular production of an RNA aptamer by ribonuclease-free marine bacteria harboring engineered plasmids: a proposal for industrial RNA drug production.

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs.

Distribution of Phototrophic Purple Nonsulfur Bacteria in Massive Blooms in Coastal and Wastewater Ditch Environments.

The biodiversity of phototrophic purple nonsulfur bacteria (PNSB) in comparison with purple sulfur bacteria (PSB) in colored blooms and microbial mats that developed in coastal mudflats and pools and wastewater ditches was investigated. For this, a combination of photopigment and quinone profiling, pufM gene-targeted quantitative PCR, and pufM gene clone library analysis was used in addition to conventional microscopic and cultivation methods. Red and pink blooms in the coastal environments contained PSB as the major populations, and smaller but significant densities of PNSB, with members of Rhodovulum predominating. On the other hand, red-pink blooms and mats in the wastewater ditches exclusively yielded PNSB, with Rhodobacter, Rhodopseudomonas, and/or Pararhodospirillum as the major constituents. The important environmental factors affecting PNSB populations were organic matter and sulfide concentrations and oxidation‒reduction potential (ORP). Namely, light-exposed, sulfide-deficient water bodies with high-strength organic matter and in a limited range of ORP provide favorable conditions for the massive growth of PNSB over co-existing PSB. We also report high-quality genome sequences of Rhodovulum sp. strain MB263, previously isolated from a pink mudflat, and Rhodovulum sulfidophilum DSM 1374T, which would enhance our understanding of how PNSB respond to various environmental factors in the natural ecosystem.

Characterization of extracellular DNA production and flocculation of the marine photosynthetic bacterium Rhodovulum sulfidophilum.

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids involved in its flocculation. Previously, we showed that the RNA fraction of these extracellular nucleic acids released into the culture medium contains mainly non-aminoacylated fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Here, we report the characterization of extracellular DNA itself and its production during cultivation. No differences were detected in nucleotide sequence between the intracellular DNA and extracellular soluble DNA on Southern blotting. Whole intracellular DNA seemed to be released from the cell. The bacterial floc was degraded by deoxyribonuclease or ribonuclease treatment, indicating that at least the extracellular DNA and RNAs in the floc are involved in the maintenance of the floc. When cultivated in nutritionally rich medium, the bacteria formed small flocs and produced large amounts of extracellular DNA, which were solubilized in the medium. In nutritionally poor medium, however, huge flocs of cells appeared and almost no extracellular soluble DNA was observed in the medium. As the floc was degraded by deoxyribonuclease treatment, it seems likely that the extracellular soluble DNA observed in the rich medium may be incorporated into the large floc and play a role in floc maintenance in poor medium. Addition of an inhibitor of quorum sensing, alpha-cyclodextrin, inhibited huge floc maintenance in the nutritionally poor medium. In the presence of alpha-cyclodextrin, the floc was rapidly degraded and extracellular soluble DNA production increased.

Extracellular tRNAs of the marine photosynthetic bacterium Rhodovulum sulfidophilum are not aminoacylated.

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces nucleic acids extracellularly. We have identified these extracellular RNAs as fully mature sized tRNAs and fragments of 16S and 23S rRNAs. Most of the tRNAs have mature 3'-terminal CCA sequences. In the present study we found that these extracellular tRNAs were not aminoacylated, although almost all intracellular tRNAs are aminoacylated.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described.

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium Rhodovulum sulfidophilum.

The marine bacterium Rhodovulum sulfidophilum is a nonsulfur phototrophic bacterium, which is known to produce extracellular nucleic acids in soluble form in culture medium. In the present paper, constructing the response regulator ctrA-deficient mutant of R. sulfidophilum, we found that this mutation causes a significant decrease in the extracellular DNA production. However, by the introduction of a plasmid containing the wild type ctrA gene into the mutant, the amount of extracellular DNA produced was recovered. This is the first and clear evidence that the extracellular DNA production is actively controlled by the CtrA in R. sulfidophilum.

Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction.

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA(Gly1) from E. coli as variable substrate (Ki 0.960 microM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA(Gly1) from E. coli (Ki 1.90 microM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA(Gly1) from E. coli followed non-competitive kinetics (Ki 4.1 microM). The porphyrins bound directly to E. coli tRNAVal, E. coli pre-tRNAGly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (microM) against E. coli tRNAVal and E. coli pre-tRNAGly were: meso-tetrakis(N-methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio)phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl)porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong pi-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.

Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum.

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs.

Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium.

Rhodovulum sulfidophilum DSM 2351 is the nonsulfur photosynthetic bacterium that efficiently releases nucleic acids into the extracellular milieu, which leads to flocculation. In this study, we determined the complete genome sequence of R. sulfidophilum DSM 2351, which will provide new insights into the mechanism of its unique nucleic acid release.

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation.

To explore the variation of the light-regulated genes during complementary chromatic acclimation (CCA), we determined the complete genome sequence of the cyanobacterium Geminocystis sp. strain NIES-3708. Within the light-regulated operon for CCA, we found genes for phycoerythrin but not phycocyanin, suggesting that this cyanobacterium modulates phycoerythrin composition only (type II CCA).

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome.

The cyanobacterium Geminocystis sp. strain NIES-3709 accumulates a larger amount of phycoerythrin than the related NIES-3708 strain does. Here, we determined the complete genome sequence of the NIES-3709 strain. Our genome data suggest that the different copy number of rod linker genes for phycoerythrin leads to the different phycoerythrin contents between the two strains.

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced.

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.