Chronicle of a Soil Bacterium: Paenibacillus polymyxa E681 as a Tiny Guardian of Plant and Human Health

The Gram-positive rhizosphere bacterium Paenibacillus polymyxa promotes plant growth and produces various antibiotics. Herein, we review research on this species over the past two and a half decades, and focus on the mechanisms of P. polymyxa strain E681, isolated from barley roots in the South Korea in 1995. Strain E681 has outstanding growth-promoting effects on barley, cucumber, pepper, sesame, and Arabidopsis thaliana and produces antimicrobial compounds that protect plants against pathogenic fungi, oomycetes, and bacteria. Induced systemic resistance elicited by treating seeds or roots with strain E681 is a possible mechanism for protecting systemic plant tissues from biotic and other environmental stresses. Genome sequencing has broadened our horizons for antibiotic development and other industrial applications beyond agricultural use. At least six gene clusters for the biosynthesis of antibiotics have been discovered, including polymyxin (pmx), which was recently re-instated as an antibiotic of last resort against Gram-negative drug-resistant bacteria. Three groups of antibiotic synthetases include the gene clusters that encode one for the non-ribosomal peptide polymyxin, fusaricidin, and tridecaptin, another for the lantibiotic paenilan, and the third for a polyketide. We successfully introduced the pmx gene cluster into the surrogate host Bacillus subtilis and created polymyxin derivatives by domain swapping. Furthermore, various E681 derivatives, including a high fusaricidin producer and strains lacking multi-antibiotics production, have been constructed by random mutagenesis and genome engineering. Thus, E681 is an important bacterium that contributes to both plant and human health

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA

Isolation of human Dna2 endonuclease and characterization of its enzymatic properties

In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase δ-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5′ and 3′ tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5′ and 3′ regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells

Activation of Protein Kinase G After Repeated Cocaine Administration Is Necessary for the Phosphorylation of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor GluA1 at Serine 831 in the Rat Nucleus Accumbens

Phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the striatum plays a crucial role in regulating the receptor-coupled signaling cascades leading to behavioral changes associated with psychostimulant exposure. The present study determined if activation of protein kinase G (PKG) contributes to the phosphorylation of AMPA receptor GluA1 subunit at the position of serine 831 (GluA1-S831) in the rat nucleus accumbens (NAc) after repeated cocaine administration. The results demonstrated that repeated intraperitoneal (i.p.) injections of cocaine (20 mg/kg) once a day for seven consecutive days significantly increased the level of phosphorylated (p)GluA1-S831. This increase was decreased by the intra-NAc infusion of either the cyclic guanosine monophosphate (cGMP) analog, Rp-8-Br-PET-cGMPS (5 nmol/1 μL), or the PKG inhibitor, KT5823 (2 nmol/1 μL). Repeated cocaine administration increased PKG binding activity to GluA1. This increase in GluA1-S831 phosphorylation after repeated cocaine administration was decreased by the intra-NAc infusion of the synthetic peptide (Tat-tagged interfering peptide (Tat-GluA1-i)), that interferes with the binding of PKG to GluA1. Intra-NAc infusion of the interfering peptide also reduced the repeated cocaine-induced increase in locomotor activity. These findings suggest that activated PKG, after repeated exposure to cocaine, binds to AMPA receptor GluA1 and is required for the phosphorylation of S831, contributing to behavioral changes

Valproic Acid Downregulates the Expression of MGMT and Sensitizes Temozolomide-Resistant Glioma Cells

Temozolomide (TMZ) has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. Valproic acid (VPA) has emerged as an anticancer drug via inhibition of histone deacetylases (HDACs), but the therapeutic advantages of a combination with VPA and TMZ remain poorly understood. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of VPA and TMZ, especially in TMZ-resistant cell lines. A combination of VPA and TMZ had a significantly enhanced antitumor effect in TMZ-resistant malignant glioma cells (T98 and U138). This enhanced antitumor effect correlated with VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, which plays an important role in cellular resistance to alkylating agents. In vitro, the combination of these drugs enhanced the apoptotic and autophagic cell death, as well as suppressed the migratory activities in TMZ-resistant cell lines. Furthermore, in vivo efficacy experiment showed that treatment of combination of VPA and TMZ significantly inhibited tumor growth compared with the monotherapy groups of mice. These results suggest that the clinical efficacy of TMZ chemotherapy in TMZ-resistant malignant glioma may be improved by combination with VPA

In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing

The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNA-dependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2Δ405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperature-sensitive growth defect of dna2Δ405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Δ405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki fragment processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the processing of Okazaki fragments, leading to genomic instability