The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for >90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.
α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. kcat/Km values of AgΔUCD and Agl-KA were 11.4 and 11.1 s–1 mg–1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.
Acetate is a significant intermediate of anaerobic fermentation. There are two pathways for converting acetate to CH4 and CO2: acetoclastic methanogenesis by acetoclastic methanogens, and syntrophic acetate oxidation by acetate-oxidizing bacteria (AOB) and hydrogenotrophic methanogens. Detailed investigations of syntrophic acetate-oxidizing bacteria (SAOB) should contribute to the elucidation of the microbial mechanisms of methanogenesis. In this study, we investigated the major phylogenetic groups of acetate-utilizing bacteria (AUB) in a mesophilic methanogenic chemostat fed with acetate as the sole carbon source by using DNA stable isotope probing (SIP) technology. The results indicated that acetoclastic methanogenesis and acetate oxidization/hydrogenotrophic methanogenesis coexisted in the mesophilic chemostat fed with acetate, operated at a dilution rate of 0.1 d–1. OTU Ace13(9-17) (KU869530), Ace13(9-4) (KU667241), and Ace13(9-23) (KU667236), assigned to the phyla Firmicutes and Bacteroidetes, were probably potential SAOB in the chemostat, which needs further investigation. Species in the phyla Proteobacteria, Deferribacteres, Acidobacteria, Spirochaetes and Actinobacteria were probably capable of utilizing acetate for their growth. Methanoculleus was likely to be the preferred hydrogenotrophic methanogen for syntrophy with AOB in the chemostat.
Coastal ecosystems, play critical ecological roles of which tidal flats are a significant component of coastal wetlands, such as habitat and nutrient cycling in aquatic biology. Microbial communities in tidal flats are known to play vital roles of self-purification. And the microbial ecology of the sediment is easily affected by human activities and pollution. In this paper, we applied pyrosequencing technology to investigate microbial communities in three different tidal flats (Ganghwa Island, Ongnyeon land region and Yeongjong Island) on the Incheon, Korea peninsula. A total of 16,906 sequences were obtained. We used these sequences to identify the dominant phyla in the three tidal flats: Proteobacteria, Chloroflexi, Actinobacteria, and Bacteroidetes. The composition of the bacterial community of Ganghwa Island and the Ongnyeon region were more similar to each other than they were to the bacterial community of Yeongjong Island. Simpson's dominance index of Yeongjong Island was higher than that of the other regions, and the Shannon diversity index of this region was the lowest. Previous research of samples in these regions indicated that the three tidal flats had similar geochemical characteristics. However, their bacterial communities were rather distinct. This might be because the analysis of microbial communities and physiochemical analysis have different perspectives. Therefore, the pyrosequencing of a bacterial community with physiochemical analysis is recommended as an effective monitoring tool for the comprehensive management of tidal flats.
Sirtuin is an NAD+-dependent histone deacetylase that is highly conserved among prokaryotes and eukaryotes. Sirtuin deacetylates histones and non-histone proteins, and it is involved in fungal growth and secondary metabolite production. Here, we screened 579 fungal culture extracts that inhibited the histone deacetylase activity of Sirtuin A (SirA), produced by the fungus Aspergillus nidulans. Eight fungal strains containing three Ascomycota, two Basidiomycota and three Deuteromycetes produced SirA inhibitors. We purified the SirA inhibitor from the culture broth of Didymobotryum rigidum JCM 8837, and identified it as 5-methylmellein—a known polyketide. This polyketide and its structurally-related compound, mellein, inhibited SirA activity with IC50 of 120 and 160 μM, respectively. Adding 5-methylmellein to A. nidulans cultures increased secondary metabolite production in the medium. The metabolite profiles were different from those obtained by adding other sirtuin inhibitors nicotinamide and sirtinol to the culture. These results indicated that 5-methylmellein modulates fungal secondary metabolism, and is a potential tool for screening novel compounds derived from fungi.
Sorbitol-6-phosphatase (EC 22.214.171.124) catalyzes sorbitol production from sorbitol-6-phosphate in certain organisms, but has not been identified unequivocally. We screened the activity of the haloacid dehalogenase-like hydrolases (HAD) superfamily and identified four HAD proteins from Escherichia coli as sorbitol-6-phosphatase. Of these proteins, HAD2 (YfbT) exhibited catalytic activity (kcat/Km) that was better than that of the previously reported "preferred" substrate. HAD1 (YniC) and HAD2 exhibited higher sorbitol-6-phosphatase activity than that of HAD12 (YbiV) and HAD13 (YidA). Therefore, genes of HAD may be useful for metabolic engineering of effective sorbitol production.
Chlamydia pneumoniae is an obligate intracellular pathogen responsible for respiratory diseases, including pneumonia and bronchitis, and is highly involved in chronic diseases, including atherosclerosis, asthma, and Alzheimer's disease. We previously showed that the host apoptotic factor caspase-9 played a crucial role for chlamydial multiplication and host apoptosis inhibition by chlamydial infection. To identify chlamydial genes interacting with human caspase-9, yeast two-hybrid screening was performed and 5 chlamydial genes, including Cpj0838 and pmpG were isolated from the C. pneumoniae genomic library. Pull-down experiments showed that caspase-9 physically bound to the Cpj0838 product and chlamydial cells, which contain PmpG proteins. This study could provide a clue to understanding host-Chlamydia interactions, especially the apoptosis repression by Chlamydia infection.