Paramixta manurensis gen. nov., sp. nov., a novel member of the family Erwiniaceae producing indole-3-acetic acid isolated from mushroom compost.

There are numerous species in the Erwiniaceae family that are important for agricultural and clinical purposes. Here we described the Erwiniaceae bacterium PD-1 isolated from mushroom (Pleurotus eryngii) compost. Comparative genomic and phylogenetic analyses showed that the strain PD-1 was assigned to a new genus and species, Paramixta manurensis gen. nov., sp. nov. in the family Erwiniaceae. From the average amino acid index, we identified the five AroBEKAC proteins in the shikimate pathway as a minimal set of molecular markers to reconstruct the phylogenetic tree of the Erwiniaceae species. The strain PD-1 containing annotated genes for ubiquinone and menaquinone produced a higher level of ubiquinone (Q8) than demethylmenaquinone (DMK8) and menaquinone (MK8) in anaerobic condition compared to aerobic condition, as similarly did the reference strains from the genera Mixta and Erwinia. Results from fatty acid methyl ester and numerical analyses of strain PD-1 showed a similarity to species of the genera Mixta and Winslowiella. This study revealed that the strain's ability to utilize polyols, such as glycerol, erythritol, and D-arabitol, distinguished the strain PD-1 from the nearest relative and other type strains. The analyzed genetic markers and biochemical properties of the strain PD-1 suggest its potential role in the process of mushroom compost through the degradation of carbohydrates and polysaccharides derived from fungi and plants. Additionally, it can produce a high concentration of indole-3-acetic acid as a plant growth-promoting agent.

Long-term efficacy of everolimus as de novo immunosuppressant on the cardiac allograft vasculopathy in heart transplant recipients.

BACKGROUND AND AIMS: Data on the long-term effects of everolimus (EVL) on the de novo immunosuppression of heart transplant (HT) recipients with progressive cardiac allograft vasculopathy (CAV) and vascular remodeling are lacking. Hence, in this study, we aimed to determine the long-term safety and efficacy of EVL as a de novo immunosuppressant therapy for CAV progression and the clinical outcomes after HT. METHODS: We retrospectively reviewed the medical records of 144 HT recipients who survived for at least one year after HT. CAV progression was assessed via serial coronary intravascular ultrasonography (IVUS) in recipients who underwent at least two IVUS studies. RESULTS: A significant attenuation in the percentage of the atheroma volume progression was observed in those who took EVL (1.2%) compared with those who took cyclosporin (CSA; 7.3%; p = 0.005 vs. EVL) or tacrolimus (TAC; 6.6%; p = 0.0052 vs. EVL) at 1 year after HT. This trend persisted for the next 3 and 5 years after HT. Moreover, the remodeling index was greater in the EVL (1.08) group than in the CSA (0.23) or TAC (-0.25) groups 1 year after HT. The results of the Kaplan-Meier analysis over a median follow-up period of 8 years revealed that there was no statistical difference in the primary endpoint between the three groups. CONCLUSIONS: De novo immunosuppression with EVL is associated with attenuated CAV progression for the first 5 years of follow-up via IVUS. Moreover, EVL has comparable long-term clinical outcomes to those of CSA- or TAC-based protocols.

Analysis of phylogenetic markers for classification of a hydrogen peroxide producing Streptococcus oralis isolated from saliva by a newly devised differential medium.

Hydrogen peroxide (H2O2) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of H2O2-producing streptococci in oral microbiota has not been setup. Here we show that o-dianisidine dye and horseradish peroxidase were useful in tryptic soy agar medium to detect and isolate H2O2-producing bacteria with the detection limit of one target colony in > 106 colony-forming units. As a proof, we isolated the strain HP01 (KCTC 21190) from a saliva sample using the medium and analyzed its characteristics. Further tests showed that the strain HP01 belongs to Streptococcus oralis in the Mitis group and characteristically forms short-chain streptococcal cells with a high capacity of acid tolerance and biofilm formation. The genome analysis revealed divergence of the strain HP01 from the type strains of S. oralis. They showed distinctive phylogenetic distances in their ROS-scavenging proteins, including superoxide dismutase SodA, thioredoxin TrxA, thioredoxin reductase TrxB, thioredoxin-like protein YtpP, and glutaredoxin-like protein NrdH, as well as a large number of antimicrobial resistance genes and horizontally transferred genes. The concatenated ROS-scavenging protein sequence can be used to identify and evaluate Streptococcus species and subspecies based on phylogenetic analysis.

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain.

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle-dependent regulation of nuclear morphology at the molecular level.

Regulatory role of cysteines in (2R, 3R)-butanediol dehydrogenase BdhA of Bacillus velezensis strain GH1-13.

Bacillus velezensis strain GH1-13 contains a (2R,3R)-butanediol dehydrogenase (R-BDH) BdhA which converts acetoin to R-BD reversibly, however, little is known about its regulatory cysteine and biological significance. We performed site-directed mutation of three cysteines in BdhA. The C37S mutant had no enzyme activity and the C34S and C177S mutants differed from each other and wild type (WT). After zinc affinity chromatography, 1 mM ZnCl2 treatment resulted in a 3-fold enhancement of the WT activity, but reduced activity of the C34S mutant by more than 2 folds compared to the untreated ones. However, ZnCl2 treatment did not affect the activity of the C177S mutant. Most of the double and triple mutant proteins (C34S/C37S, C34S/C177S, C37S/C177S, and C34S/C37S/C177S) were aggregated in zinc resins, likely due to the decreased protein stability. All of the purified WT and single mutant proteins increased multiple intermolecular disulfide bonds in the presence of H2O2 as the buffer pH decreased from 7.5 to 5.5, whereas an intramolecular disulfide bond of cysteine 177 and another cysteine in the CGIC motif region was likely formed at pH higher than pKa of 7.5. When pH varied, WT and its C34S or C177S mutants reduced acetoin to R-BD at the optimum pH 5.5 and oxidized R-BD to acetoin at the optimum pH 10. This study demonstrated that cysteine residues in BdhA play a regulatory role for the production of acetoin and R-BD depending on pH as well as metal binding and oxidative stress.

A native conjugative plasmid confers potential selective advantages to plant growth-promoting Bacillus velezensis strain GH1-13.

The conjugative plasmid (pBV71) possibly confers a selective advantage to Bacillus velezensis strain GH1-13, although a selective marker gene is yet to be identified. Here we show that few non-mucoid wild-type GH1-13 cells are spontaneously converted to mucoid variants with or without the loss of pBV71. Mucoid phenotypes, which contain or lack the plasmid, become sensitive to bacitracin, gramicidin, selenite, and tellurite. Using the differences in antibiotic resistance and phenotype, we isolated a reverse complement (COM) and a transconjugant of strain FZB42 with the native pBV71. Transformed COM and FZB42p cells were similar to the wild-type strain GH1-13 with high antibiotic resistance and slow growth rates on lactose compared to those of mucoid phenotypes. RT-PCR analysis revealed that the expression of plasmid-encoded orphan aspartate phosphatase (pRapD) was coordinated with a new quorum-sensing (QS) cassette of RapF2-PhrF2 present in the chromosome of strain GH1-13, but not in strain FZB42. Multi-omics analysis on wild-type and plasmid-cured cells of strain GH1-13 suggested that the conjugative plasmid expression has a crucial role in induction of early envelope stress response that promotes cell morphogenesis, biofilm formation, catabolite repression, and biosynthesis of extracellular-matrix components and antibiotics for protection of host cell during exponential phase.

Endoribonuclease-mediated control of hns mRNA stability constitutes a key regulatory pathway for Salmonella Typhimurium pathogenicity island 1 expression.

Bacteria utilize endoribonuclease-mediated RNA processing and decay to rapidly adapt to environmental changes. Here, we report that the modulation of hns mRNA stability by the endoribonuclease RNase G plays a key role in Salmonella Typhimurium pathogenicity. We found that RNase G determines the half-life of hns mRNA by cleaving its 5' untranslated region and that altering its cleavage sites by genome editing stabilizes hns mRNA, thus decreasing S. Typhimurium virulence in mice. Under anaerobic conditions, the FNR-mediated transcriptional repression of rnc encoding RNase III, which degrades rng mRNA, and simultaneous induction of rng transcription resulted in rapid hns mRNA degradation, leading to the derepression of genes involved in the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Together, our findings show that RNase III and RNase G levels-mediated control of hns mRNA abundance acts as a regulatory pathway upstream of a complex feed-forward loop for SPI-1 expression.

Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku.

The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.

Structural basis for lamin assembly at the molecular level.

Nuclear structure and function are governed by lamins, which are intermediate filaments that mostly consist of α-helices. Different lamin assembly models have been proposed based on low resolution and fragmented structures. However, their assembly mechanisms are still poorly understood at the molecular level. Here, we present the crystal structure of a long human lamin fragment at 3.2 Å resolution that allows the visualization of the features of the full-length protein. The structure shows an anti-parallel arrangement of the two coiled-coil dimers, which is important for the assembly process. We further discover an interaction between the lamin dimers by using chemical cross-linking and mass spectrometry analysis. Based on these two interactions, we propose a molecular mechanism for lamin assembly that is in agreement with a recent model representing the native state and could explain pathological mutations. Our findings also provide the molecular basis for assembly mechanisms of other intermediate filaments.

Applications of different solvents and conditions for differential extraction of lipopolysaccharide in Gram-negative bacteria.

Lipopolysaccharide (LPS) is one of the major components in the outer membrane of Gram-negative bacteria. However, its heterogeneity and variability in different bacteria and differentiation conditions make it difficult to extract all of the structural variants. We designed a solution to improve quality and biological activity of LPS extracted from various bacteria with different types of LPS, as compared to conventional methods. We introduced a quality index as a simple measure of LPS purity in terms of a degree of polysaccharide content detected by absorbance at 204 nm. Further experiments using gel electrophoresis, endotoxin test, and macrophage activation test were performed to evaluate the performance and reliability of a proposed 'T-sol' method and the biological effectiveness and character of the LPS products. We presented that the T-sol method had differential effects on extraction of a RAW 264.7 cell-activating LPS, which was effective in the macrophage activation with similar effects in stimulating the production of TNF-alpha. In conclusion, the T-sol method provides a simple way to improve quality and biological activity of LPS with high yield.

Plant growth-promoting activity of beta-propeller protein YxaL secreted from Bacillus velezensis strain GH1-13.

YxaL is conserved within the Bacillus subtilis species complex associated with plants and soil. The mature YxaL protein contains a repeated beta-propeller domain, but the subcellular location and function of YxaL has not been determined. The gene encoding the mature YxaL protein was PCR amplified from genomic DNA of B. velezensis strain GH1-13 and used for recombinant protein production. A rabbit polyclonal antibody against the purified YxaL was generated and used for western blotting to determine the constitutive expression and secretion of YxaL. During normal culture growth of strain GH1-13, levels of the constitutively secreted YxaL were slowly rising to 100 µg L-1, and degraded with a half-life of 1.6 h in the culture medium. When the effects of YxaL on plant seed germination and seedling growth were examined, it was shown that seed treatment of Arabidopsis thaliana and rice (Oryza sativa L.) with purified YxaL at the optimal concentration of 1 mg L-1 was effective at improving the root growth of plants. Seedlings from the treated Arabidopsis seeds markedly increased transcription of a 1-aminocyclopropane-1-carboxylate synthetase marker gene (ACS11) but reduced expression of auxin- and abscisic acid-responsive marker genes (IAA1, GH3.3, and ABF4), especially when provided with exogenous auxin. Horticulture experiments showed that pepper (Capsicum annuum) seeds treated with 1 mg L-1 YxaL in a soaking solution increased shoot growth and improved tolerance to drought stress. We hypothesize that YxaL secreted from plant growth-promoting Bacillus cells has a significant impact on plant roots, with the potential to improve plant growth and stress tolerance.

Divergent rRNAs as regulators of gene expression at the ribosome level.

It is generally assumed that each organism has evolved to possess a unique ribosomal RNA (rRNA) species optimal for its physiological needs. However, some organisms express divergent rRNAs, the functional roles of which remain unknown. Here, we show that ribosomes containing the most variable rRNAs, encoded by the rrnI operon (herein designated as I-ribosomes), direct the preferential translation of a subset of mRNAs in Vibrio vulnificus, enabling the rapid adaptation of bacteria to temperature and nutrient shifts. In addition, genetic and functional analyses of I-ribosomes and target mRNAs suggest that both I-ribosomal subunits are required for the preferential translation of specific mRNAs, the Shine-Dalgarno sequences of which do not play a critical role in I-ribosome binding. This study identifies genome-encoded divergent rRNAs as regulators of gene expression at the ribosome level, providing an additional level of regulation of gene expression in bacteria in response to environmental changes.

A mitochondrial proteome profile indicative of type 2 diabetes mellitus in skeletal muscles.

The pathogenesis of type 2 diabetes mellitus (T2DM) is closely associated with mitochondrial functions in insulin-responsive tissues. The mitochondrial proteome, compared with the mitochondrial genome, which only contains 37 genes in humans, can provide more comprehensive information for thousands of mitochondrial proteins regarding T2DM-associated mitochondrial functions. However, T2DM-associated protein signatures in insulin-responsive tissues are still unclear. Here, we performed extensive proteome profiling of mitochondria from skeletal muscles in nine T2DM patients and nine nondiabetic controls. A comparison of the mitochondrial proteomes identified 335 differentially expressed proteins (DEPs) between T2DM and nondiabetic samples. Functional and network analyses of the DEPs showed that mitochondrial metabolic processes were downregulated and mitochondria-associated ER membrane (MAM) processes were upregulated. Of the DEPs, we selected two (NDUFS3 and COX2) for downregulated oxidative phosphorylation and three (CALR, SORT, and RAB1A) for upregulated calcium and protein transport as representative mitochondrial and MAM processes, respectively, and then confirmed their differential expression in independent mouse and human samples. Therefore, we propose that these five proteins be used as a potential protein profile that is indicative of the dysregulation of mitochondrial functions in T2DM, representing downregulated oxidative phosphorylation and upregulated MAM functions.

Minimum InDel pattern analysis of the Zika virus.

BACKGROUND: The Zika virus (ZIKV) can cause microcephaly and congenital abnormalities in the foetus. Recent studies have provided insights into the evolution of ZIKV from the current and previous outbreaks, but the types have not been determined. RESULTS: We analysed the insertions and deletions (InDels) in 212 ZIKV polyproteins and 5 Dengue virus (DENV) reference sequences. Spearman correlation tests for the minimum InDel (minInDel) patterns were used to assess the type of polyprotein. Using the minInDel frequencies calculated from polyproteins with 11 elements, likelihood estimation was conducted to correct the evolutionary distance. The minInDel-corrected tree topology clearly distinguished between the ZIKV types (I and II) with a unique minInDel character in the E protein. From the 10-year average genetic distance, the African and Asian lineages of ZIKV-II were estimated to have occurred ~ 270 years ago, which is unlikely for ZIKV-I. CONCLUSIONS: The minInDel pattern analysis showed that the minInDel in the E protein is targetable for the rapid detection and determination of the virus types.

Analysis of endogenous lipids during intestinal wound healing.

Intestinal wound healing is a new therapeutic goal for inflammatory bowel disease (IBD) as complete healing of the mucosa is the key element of clinical remission in IBD. Previous studies showed that termination of inflammation can be achieved by adding pro-resolving lipids like DHA and EPA exogenously. However, the roles of these lipids in mucosal healing have not been investigated. To recapitulate intestinal healing process, mice were received dextran sodium sulfate (DSS) for 7 days in the drinking water followed by regular tap water for 5 additional days. DSS-induced intestinal inflammation featuring body weight loss, histological tissue damage, increased cytokine production and infiltration of inflammatory cells was gradually reduced upon switching to water. To investigate whether endogenous lipids play a role in mucosal healing, the lipidomics analysis of mouse serum was performed. Reduced levels of arachidonic acid, the biosynthetic precursor of prostaglandin F (PGF)2α, 19H-PGF1α, the metabolite of prostacyclin, and 20H-PGF2α, the metabolite of PGF2α, suggest subsiding inflammation. In contrast, increased levels of an active metabolite of resolvin D1 along with decreased levels of its precursor DHA as well as decreased levels of the precursor of resolvin E, 18-hydroxy-eicosapentaenoic acid, suggest inauguration of mucosal healing by endogenous lipids. Furthermore, exogenously supplied fish oil enhanced the process even further. These results suggest the presence of mucosal healing regulated by endogenous pro-healing lipids and also indicate that the remission state of IBD could be prolonged by enhancing the levels of these lipids.

Determination of the binding site of 2-aminothiazole derivative with importin ß1 by UV-crosslinking experiment.

Importin ß1 (KPBN1) appears to be overexpressed in several cancer cells and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, but do not affect normal cells. These results indicate that KPNB1 is a potential target and inhibition of KPNB1 can be used as a novel therapeutic approach for the treatment of cancer. Recently, we identified the aminothiazole derivative 1 as a KPNB1-targeted anticancer agent. Herein, we report that compound 1 binds strongly to KPNB1, in a pocket centered around serine-476, as shown by UV-crosslinking and tandem mass spectrometry experiments, and supported using a model derived from molecular docking.

Identification of KPNB1 as a Cellular Target of Aminothiazole Derivatives with Anticancer Activity.

We found that aminothiazole derivative (E)-N-(5-benzylthiazol-2-yl)-3-(furan-2-yl)acrylamide (1) has strong anticancer activity, and undertook proteomics approaches to identify the target protein of compound 1, importin ß1 (KPNB1). A competitive binding assay using fluorescein-labeled 1 showed that 1 has strong binding affinity for KPNB1 (Kd : ∼20 nm). Furthermore, through western blotting assays for KPNB1, KPNA2, EGFR, ErbB2, and STAT3, we confirmed that 1 has inhibitory effects on the importin pathway. KPBN1 appears to be overexpressed in several cancer cells, and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, while leaving non-cancerous cells unaffected. Therefore, compound 1 is a promising new lead for the development of KPNB1-targeted anticancer agents. Fluorescein-labeled 1 could be a useful quantitative probe for the development of novel KPNB1 inhibitors.

Functional Analysis of Vibrio vulnificus Orthologs of Escherichia coli RraA and RNase E.

RNase E plays an important role in the degradation and processing of RNA in Escherichia coli. The enzymatic activity of RNase E is controlled by the protein inhibitors RraA and RraB. The marine pathogenic bacterium Vibrio vulnificus also contains homologs of RNase E and RraA, designated as RNase EV, RraAV1, and RraAV2. Here, we report that RraAV1 actively inhibits the enzymatic activity of RNase EV in vivo and in vitro by interacting with the C-terminal domain of RNase EV. Coexpression of RraAV1 reduced ribonucleolytic activity in the cells overproducing RNase EV and consequently restored normal growth of these cells. An in vitro cleavage assay further demonstrated that RraAV1 efficiently inhibits the ribonucleolytic activity of RNase EV on BR10 + hpT, a synthetic oligonucleotide containing the RNase E cleavage site of RNA I. Our findings suggest that RraAV1 plays an active role in RNase EV-mediated RNA cleavage in V. vulnificus.

Conditional probability analysis of multidrug resistance in Gram-negative bacilli isolated from tertiary medical institutions in South Korea during 1999-2009.

Multidrug resistance of Gram-negative bacilli is a major problem globally. However, little is known about the combined probability of resistance to various antibiotics. In this study, minimum inhibitory concentrations of widely used antibiotics were determined using clinical isolates of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, randomly chosen from strain collections created during 1999-2009 in tertiary medical institutions in Seoul, South Korea. To analyze combined efficacy of antibiotics against a subgroup of isolates, conditional probabilities were determined based on arbitrary, non-independent patterns of antimicrobial susceptibility and resistance. Multidrug resistance, defined as resistance to three or more classes of antibiotics, was observed in the following order: A. baumannii (96%), P. aeruginosa (65%), E. coli (52%), and K. pneumoniae (7%). A. baumannii strains resistant to gentamicin were found to be resistant to a number of antibiotics, except for colistin and polymyxin B. Resistance to gentamicin following exposure to this antibiotic was highly likely to lead to multidrug resistance in all four microbes. This study shows a causal relationship between gentamicin resistance and the prevalence of multidrug resistance in clinical isolates of Gramnegative bacilli in South Korea during 1999-2009 and suggests the importance of prudent use of gentamicin in hospitals.