Increased tolerance to light stress in cyanobacteria is a desirable feature for their applications. Here, we obtained a high light tolerant (Tol) strain of Synechocystis sp. PCC6803 through an adaptive laboratory evolution, in which the cells were repeatedly sub-cultured for 52 days under high light stress conditions (7000 to 9000 micromol m(-2) s(-1)). Although the growth of the parental strain almost stopped when exposed to 9000 micromol m(-2) s(-1), no growth inhibition was observed in the Tol strain. Excitation-energy flow was affected because of photosystem II damage in the parental strain under high light conditions, whereas the damage was alleviated and normal energy flow was maintained in the Tol strain. The transcriptome data indicated an increase in isiA expression in the Tol strain under high light conditions. Whole genome sequence analysis and reverse engineering revealed two mutations in hik26 and slr1916 involved in high light stress tolerance in the Tol strain.
Title: Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass Uraji M, Tamura H, Mizohata E, Arima J, Wan K, Ogawa K, Inoue T, Hatanaka T Ref: Applied Environmental Microbiology, 84:, 2018 : PubMed
Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 A to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.
In this study, the regional rat brain distribution of radioiodinated o-iodo-trans-decalinvesamicol ([(125) I]OIDV) was determined in vivo to evaluate its potential as a single-photon emission computed tomography (SPECT) imaging probe for vesicular acetylcholine transporter (VAChT). Following intravenous injection, [(125) I]OIDV passed freely across the blood-brain barrier and accumulated in rat brain. The accumulation of [(125) I]OIDV in rat brain was significantly reduced by coadministration of (+/-)-vesamicol (0.125 micromol). In contrast, the coadministration of sigma-receptor ligands, such as (+)-pentazocine (0.125 micromol) as a sigma-1 receptor ligand and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (0.125 micromol) as a sigma-1 and sigma-2 receptor ligands, barely affected the accumulation of [(125) I]OIDV in rat brain. These findings in vivo were corroborated by autoradiographic analysis ex vivo. The authors found that the tracer binds with pharmacological selectivity to VAChT in rat brain and predicted that it may likewise serve in translational SPECT imaging studies of this marker in the integrity of cholinergic innervations. Synapse 68:107-113, 2014. (c) 2013 Wiley Periodicals, Inc.
Mycobacterium avium complex (MAC) infection causes disseminated disease in immunocompromised hosts, such as human immunodeficiency virus (HIV)-positive patients, and pulmonary disease in persons without systemic immunosuppression, which has been increasing in many countries. In Japan, the incidence of pulmonary MAC disease caused by M. avium is about 7 times higher than that caused by M. intracellulare. To explore the bacterial factors that affect the pathological state of MAC disease caused by M. avium, we determined the complete genome sequence of the previously unreported M. avium subsp. hominissuis strain TH135 isolated from a HIV-negative patient with pulmonary MAC disease and compared it with the known genomic sequence of M. avium strain 104 derived from an acquired immunodeficiency syndrome patient with MAC disease. The genome of strain TH135 consists of a 4,951,217-bp circular chromosome with 4,636 coding sequences. Comparative analysis revealed that 4,012 genes are shared between the two strains, and strains TH135 and 104 have 624 and 1,108 unique genes, respectively. Many strain-specific regions including virulence-associated genes were found in genomes of both strains, and except for some regions, the G+C content in the specific regions was low compared with the mean G+C content of the corresponding chromosome. Screening of clinical isolates for genes located in the strain-specific regions revealed that the detection rates of strain TH135-specific genes were relatively high in specimens isolated from pulmonary MAC disease patients, while, those of strain 104-specific genes were relatively high in those from HIV-positive patients. Collectively, M. avium strains that cause pulmonary and disseminated disease possess genetically distinct features, and it suggests that the acquisition of specific genes during strain evolution has played an important role in the pathological manifestations of MAC disease.
The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL. HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity.
Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6.
Title: The glutamate receptor agonist, AMPA, induces acetylcholine release in guinea pig cochlea; a microdialysis study Hoya N, Ogawa K, Inoue Y, Takiguchi Y, Kanzaki J Ref: Neuroscience Letters, 311:206, 2001 : PubMed
Acetylcholine (Ach) has been considered a major neurotransmitter in the inner ear efferent nerve endings. A bioassay analysis has shown that the electrical stimulation of the crossed olivocochlear bundle increased the Ach-like activity in the perilymph. Applying in vivo microdialysis techniques and high-performance liquid chromatography to the perilymph, the change of Ach level was thus measured before and after alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), a glutamate receptor agonist, was added to the perfusate. Ach was only detectable when the perfusate contained an acetylcholinesterase inhibitor. The level of Ach increased 2-3-fold immediately after AMPA was administered. Our data suggest that the afferent stimulation, such as the administration of AMPA, may therefore induce the release of Ach from the efferent nerve endings.
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
