An Indonesian soil fungus, Talaromyces pinophilus BioMCC-f.T.3979 was cultured to find novel scaffolds of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We obtained altenusin (1), which inhibits PfDHODH, with an IC50 value of 5.9 μM, along with other metabolites: mitorubrinol (2) and mitorubrinic acid (3). Compounds 1 and 2 inhibited PfDHODH but displayed no activity against the human orthologue. They also inhibited P. falciparum 3D7 cell growth in vitro. Compound 3 showed little PfDHODH inhibitory activity or cell growth inhibitory activity.
The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds
The genes encoding α-1,3-glucanases (Agls; AglST1 and AglST2) from Streptomyces thermodiastaticus HF3-3 were cloned and were then expressed in Escherichia coli Rosetta-gami B (DE3). We purified the resultant histidine (His)-tagged α-1,3-glucanases (recombinant enzymes, rAglST1 and rAglST2). Both the recombinant enzymes were similar to the wild-type enzymes. We examined the effects of rAglST1 and rAglST2 on the formation and degradation of biofilms on glass plates with Streptococcus mutans NRBC 13955 by evaluating the biofilm content (%), release of reducing sugar (mM), release of S. mutans (log CFU/mL), and the biofilm structure using laser scanning microscopy (LSM). The results showed that after incubation for 16 h, rAglST1 and rAglST2 reduced the formation of biofilm to 52% and 49% of the control, respectively. The result may reflect the fact that the concentration of the reducing sugar and the number of S. mutans cells in the rAglATs-added medium were higher than in the control medium. After an 8-h treatment with rAglST1 and rAglST2, biofilms decreased to less than 60% of the control. The number of S. mutans cells in the reaction mixture gradually increased during the incubation period. The enzymes can degrade the biofilms that were pre-formed on the glass plate by more than 50% after a 30-min incubation in the presence of toothpaste ingredients (1% w/v of sodium fluoride, benzethonium chloride, and sodium dodecyl sulfate) at 50°C. Our study showed that rAglST1 and rAglST2 have advantageous properties for dental care applications.
Bacillus based probiotics are becoming relevant as alternatives to antibiotics used in poultry production and in other animal husbandry. This study describes the isolation of 48 Bacillus spp. candidates, from chickens and chicken environments, for use as potential probiotics in poultry production. These isolates, plus a further 18, were tested in a comprehensive in vitro screening regime that was specifically designed to select the best isolates that satisfied multiple modes of action desirable for commercial poultry probiotics. This screening programme involved the evaluation of the ability of the isolates to survive and grow in the limiting conditions of the chicken gastrointestinal tract. Only 11 of the isolates fulfilled these criteria; hence, they were further evaluated for the ability to adhere to epithelial cells, produce extracellular enzymes, and to demonstrate antagonistic activity against selected pathogens of significant importance in poultry production. Of these, a total of 6 isolates were selected, due to their all-round probiotic capability. Identification by 16S RNA sequencing confirmed these isolates as B. subtilis and B. velezensis, identities which are generally regarded as safe. The Bacillus isolates reported in our study exhibit strong all-inclusive probiotic effects and can potentially be formulated as a probiotic preparation for poultry production.
The discharge of industrial dyes and their breakdown products is often environmentally harmful. Here, we describe a biodegradation method using Burkholderia multivorans CCA53, which exhibits a capacity to degrade azo dyes, particularly ethyl red. Under optimized culture conditions, 100 μM ethyl red was degraded more than 99% after incubation for 8 h. Real-time PCR analysis of azoR1 and azoR2, encoding two azoreductases, revealed that transcription of these genes is enhanced early during culture under optimized conditions. For a more practical approach, hydrolysates were prepared from eucalyptus or Japanese cedar chips or rice straw, and rice straw hydrolysate was used as the best medium for ethyl red biodegradation. Under those conditions, ethyl red was also degraded with high efficiency (>91%). We have thus constructed a potentially economical method for the biodegradation of ethyl red.
An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.
An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130~159 aa, L1 and 175~209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY(6x), with ATP(s), and this complex then docks through ClpQ(6x) for ultimate proteolytic degradation.
Naturally occurring fungi have been used in the traditional production of dried bonito, Katsuobushi, in Japan. In this study, we analyzed the fungal population present during Katsuobushi production. Amplicon sequence analysis of ITS1 indicated that Aspergillus spp. are predominant throughout the production process. In addition, culture-dependent analyzes identified three species Aspergillus chevalieri, Aspergillus montevidensis, and Aspergillus sydowii, based on sequencing of benA, caM, and rpb2 genes. A. chevalieri isolates were classified into teleomorphic and anamorphic strains based on morphological analysis. A. chevarieri was the dominant species throughout the production process, whereas A. montevidensis increased and A. sydowii decreased in abundance during Katsuobushi production. Our study will enhance the understanding of fungal species involved in traditional Katsuobushi production.
Cyanobacteria are an important component in the rice field ecosystem and are a well known source of natural biofertilizer. Pesticidal application for the control of pests in rice field soil has led to several environmental problems, and poses a great threat to these beneficial microorganisms. Studies on the impact of pesticides on the diazotrophic growth and survivability of these microorganisms have recently gained much attention. The present paper describes the effects of an iterated use of the insecticide deltamethrin (2.8% EC) on the growth and nitrogen fixation capacity of the filamentous cyanobacterium Calothrix sp. (strain GUEco 1002). This organism has shown a varying degree of sensitivity to the insecticide. For evaluating the deltamethrin toxicity, the test organism was subjected to varying concentrations of deltamethrin i.e. 17.5 ppm, 35 ppm, 70 ppm and 140 ppm based upon LC50 for 20 days. The data obtained in the laboratory revealed that the treatment of the test organism with deltamethrin (17.5–140 ppm) negatively affected its growth, pigments, protein and nitrogen content in a time dose dependent manner. In contrast, carbohydrate content significantly increased with increasing concentrations of deltamethrin, this effect being more prominent at 140 ppm treatment (38%). At this high level (140 ppm), the test organism showed a significant decrease in dry weight biomass (46%), chlorophyll-a (72%), carotenoids (57%), phycocyanin (67%), protein (69%) and nitrogen content (61%) over the control. A little, but insignificant, stimulatory effect on nitrogen content was recorded at 17.5 ppm of the insecticide which however, was the opposite in the case of growth, pigments, carbohydrate and protein content.
Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.
Oscillation in bacterial bioluminescence from Photobacterium kishitanii liquid culture was examined regarding reproducibility and bacterial cell activities, i.e., dissolved oxygen (DO) consumption, esterase activity, and product production rate. A frequent increase in DO was suspected to be due to a rapid decrease in luminescence, and a simple model describing not only the monotonous decrease in cell activity, but also the luminescence-DO relationship is proposed.
We recently developed an Aspergillus oryzae strain in which malonyl-coenzyme A (CoA) supply is strengthened by the deletion of snfA and SCAP as an efficient host to produce a plant polyketide, curcumin. Here, we examined the effectiveness of this strain in producing another polyketide, atrochrysone carboxylic acid (ACA), which is synthesized from eight molecules of malonyl-CoA using an iterative type I polyketide synthase, ACA synthase (ACAS), and atrochrysone carboxyl ACP thioesterase (ACTE) in Aspergillus terreus. When ACAS and ACTE were introduced, the A. oryzae ΔsnfAΔSCAP strain produced approximately four times more ACA-related polyketides than did the control strain expressing both genes. This result further demonstrated the availability of the A. oryzae ΔsnfAΔSCAP strain for heterologous polyketide production.
A new chaetochiversin analog, designated chaetochiversin C (1), was discovered from a cultured broth of fungal strain FKI-7792 by physicochemical screening. This strain was identified as a member of genus Neocosmospora based on morphology and DNA barcoding. The partially relative configuration of 1 was determined by 13C-NMR chemical shifts of the acetonide analog of 1. The absolute configuration was determined using an advanced Mosher's method. Compound 1 was assessed for anti-tumor, anti-microbial, and anti-malarial activities, and its ability to scavenge or quench reactive oxygen species (ROS), such as superoxide anion radicals, hydroxy radicals and singlet oxygen (1O2). Compound 1 showed a quenching effect on 1O2.
The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.
MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same. A series of biosynthetic genes for ECA, including ones for the corresponding nucleotide sugars, have been identified and extensively characterized. In this study, we found that knockouts as to the respective genes for ECA biosynthesis can grow in the minimum medium with the normal expression level of MPIase, indicating that MPIase can be biosynthesized de novo without the utilization of any compounds generated through ECA biosynthesis. Conversely, ECA was expressed normally upon MPIase depletion. From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.
Mating is a promising breeding method for industrial yeast. Although sake yeast has a low spore-formation ability, segregants exhibiting a mating type have been isolated from sake yeast K7. Here, we constructed zygotes from a cross between those segregants and a laboratory yeast strain. Because most sake and brewing yeast strains are prototrophs, we developed a PCR-based method to confirm that mating had taken place based on genome sequencing data and differences in nucleotide sequences between the two parental strains. The mated strain, termed S. cerevisiae MITOY123, showed restored spore-formation ability, unlike most sake and brewing yeast strains. By using the mated yeast strain MITOY123, it was possible to carry out tetrad analysis for the trait of the absence of off-flavour due to phenolic products such as 4-vinylguiacol (4-VG) in sake yeast K7. This tetrad analysis indicated that a single genetic region around the gene PAD1 is responsible for the absence of phenolic off-flavour in sake yeast K7. In order to aid the breeding of sake and brewing yeast strains by mating, we also identified a restriction fragment length polymorphism (RFLP) marker for the absence of phenolic off-flavour production in strains derived from sake yeast K7. Collectively, our data show that it is possible to breed new sake and brewing yeast strains by mating and to test for the absence of phenolic off-flavour production in resultant strains easily by RFLP analysis.
Red koji is produced from cultivating rice with Monascus strains that contain various types of fungal secondary metabolites, such as red pigments and monacolin K. Monascus strain also produces citrinin—a mycotoxin. In this study, Monascus purpureus KUPM5 isolated from the Thai fermented food, sufu, was mutagenized to reduce its citrinin production using UV irradiation, NTG treatment, and a combination of UV and NTG. Screening of the mutants using plate bioassay based on the inhibitory effect against Bacillus subtilis enables the selection of 10 mutants. The mutant strains KS301U and KS302U showed an 80% reduction in citrinin production in red koji compared with the wild type (wt), and maintained the ability to produce red pigments similar to the wild type. Activities of enzymes, α-amylase, protease, and lipase, from red koji extract produced by the mutant strain KS302U, were higher than those of the wt, whereas those of the mutant strain KS301U were similar to those of the wt. Consequently, strains KS301U and KS302U were successfully selected as strains suitable for producing red koji and fermented food.
A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The molecular mass of the enzyme, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, Fe3+, and Fe2+. The highest affinity exhibited by the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying its highest degrading activity towards the Aα-chains followed by Bβ chains and Cγ chains. The enzyme was also activated by plasminogen, indicating its ability to degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion into plasmin, or (b) by direct hydrolysis. It degraded thrombin, suggesting that it may act as an anticoagulant to prevent thrombosis. Taken together, our results indicate the potential of this enzyme in controlling cardiovascular disease.