Siderophores are considered to have a good potential as decontamination agents owing to their metal-chelating abilities. In order to confirm whether siderophores can be used in the recovery of metal ions, a siderophore (or metallophore) exhibiting Co2+-chelating activity was screened to demonstrate its ability to recover Co2+ from an aqueous solution. A siderophore-producing bacterium, Pandoraea sp. HCo-4B, was identified from a screen of Co2+-resistant bacteria grown in an aerobic enrichment culture with a Co2+-supplemented medium. After incubation of the crude extracted siderophore in a Co2+-containing solution, the Co2+-siderophore complex was adsorbed on to a C18 column. The bound Co2+ was eluted from the column by the addition of 10 mM H2SO4. The recovered amount of Co2+ was proportional to the amount of the added siderophore. We observed that the siderophore identified in this study binds to Co2+ at a 1:1 ratio.
This study examines the ability of the quorum-sensing molecules (QSMs) farnesol and tryptophol to induce programmed cell death of the pathogenic fungus Candida albicans, to alter the expression of apoptosis-related genes, and to reduce the pathogenicity and virulence of C. albicans in Galleria mellonella. Our results showed that both farnesol and tryptophol inhibited C. albicans germ tube formation. In the QSM-treated group, the expression levels of the apoptosis genes increased, whereas the expression level of the anti-apoptosis gene decreased. Further, pretreatment of C. albicans with tryptophol or farnesol prior to G. mellonella larval infection significantly enhanced host survival compared with larvae infected with untreated C. albicans. Thus, farnesol and tryptophol may trigger apoptosis of C. albicans in vitro and reduce the virulence of C. albicans in vivo. Although further study is needed to identify the precise mechanisms underlying the antifungal properties of farnesol and tryptophol, these results suggest that QSMs may be effective agents for controlling fungal infection.
The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml–1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 μg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 μg/ml, respectively.
Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). Sequencing and annotating the genome of this strain led to the identification of a single gene coding for the key enzyme for the degradation of rubber: the latex clearing protein (Lcp). In this study, we showed that LcpSH22a—contrary to other already characterized rubber cleaving enzymes—is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcpSH22a was heterologously expressed in an Escherichia coli strain and purified with a functional His6- or Strep-tag. Applying liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS) and a spectrophotometric pyridine hemochrome assay, heme b was identified as a cofactor. Furthermore, heme-associated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30°C and 7, respectively, were determined using an oxygen consumption assay. Cleavage of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by the oxygenase was confirmed via detection of carbonyl functional groups containing cleavage products, using Schiff's reagent and electrospray ionization mass spectrometry (ESI-MS).
The filamentous fungus Trichoderma reesei is one of the most important fungi for the production of cellulases and xylanases, which can be used for biofuel production from lignocellulose. We aimed to develop an effective selection marker system for more extensive functional genomic studies in the fungus T. reesei, and to construct better industrial transformants for producing cellulases. Here, we present a novel effective G418 selection marker to use a codon-optimized neomycin phosphotransferase II gene nptII to transform T. reesei. We developed an effective and erasable selection marker, lcNG, and a combined genetic transformation system for gene manipulation in T. reesei using a two-Agrobacterium-mediated transformation method. This transformation strategy combines two steps in the transformation protocol, which saves 15–30-day's time. The system could be a useful tool for the genetic engineering of T. reesei.
Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.
Currently, actinomycetes and myxobacteria are the only bacteria believed to form sporangia. Here, we describe a sporangium-forming process identified in Dictyobacter aurantiacus strain S27T belonging to the class Ktedonobacteria in the phylum Chloroflexi. Microscopic observations showed that strain S27T forms a substrate mycelium and subsequently produces globose or subglobose terminal sporangia arising from the vegetative mycelia through short stalk cells. This morphogenetic differentiation is similar to that seen in members of Actinoplanes belonging to the class Actinobacteria. However, unlike in Actinoplanes, motile spores could not be observed. This is the first report of the existence of a bacterium, other than actinomycetes and myxobacteira, with a complex morphogenetic differentiation that forms sporangia and is an important microbiological discovery.