Abstract
Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that ...deletion of
rfs
, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in
Mucor lusitanicus
, led to a lower virulence in diabetic mice and nematodes. Upregulation of
rfs
correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H
2
O
2
in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing
M. lusitanicus
in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (
pkaR1
), correlated with a decrease in the toxicity of SS, downregulation of
rfs
, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in
M. lusitanicus
. Moreover,
rfs
upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of
rfs
in
M. lusitanicus
infection.
The pUM505 plasmid, isolated from a clinical
isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard
strain PAO1. CIP is an antibiotic of the quinolone family that is ...used to treat
infections.
analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the
gene in pUM505 displays 40% amino acid identity to the
aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned
(renamed
, for
iprofloxacin
esistance
rotein,
lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-
, conferred resistance to CIP on
strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in
, which involves phosphorylation of the antibiotic.
The impact of Cr(VI) in sunflower roots has been studied, focusing on the oxidation of polyunsaturated fatty acids. Plants were grown hydroponically in the presence of 0, 1.0, 5.0 and 25 mgCr L−1. ...Methanolic root extracts were analyzed by capillary liquid chromatography coupled through negative electrospray ionization to a quadrupole-time of flight mass spectrometry (capHPLC-ESI-QTOF-MS). Using partial least squares algorithm, eighteen features strongly affected by Cr(VI) were detected and annotated as linoleic acid (LA), alpha-linolenic acid (ALA) and sixteen oxidation products containing hydroperoxy-, epoxy-, keto-, epoxyketo- or hydroxy-functionalities, all of them classified as oxylipins. Inspection of the MS/MS spectra acquired for features eluting at different retention times but assigned as a sole compound, confirmed isomers formation: three hydroperoxy-octadecadienoic acids (HpODE), two oxo-octadecadienoic acids (OxoODE) and four epoxyketo-octadecenoic acids (EKODE). Around 70% of metabolites in sunflower LA metabolic pathway were affected by Cr(VI) stress and additionally, four EKODE isomers not included in this pathway were found in the exposed roots. Among ALA-derived oxylipins, 13-epi-12-oxo-phytodienoic acid (OPDA) is of relevance, because of its participation in the activation of secondary metabolism. The abundances of all oxylipins were directly dependent on the Cr(VI) concentration in medium; furthermore, autooxidation of LA to HpODE isomers was observed after incubation with Cr(VI). These results point to the direct involvement of Cr(VI) in non-enzymatic oxidation of fatty acids; since oxylipins are signaling molecules important in plant defensive response, their synthesis under Cr(VI) exposure sustains the ability of sunflower to grow in Cr(VI)-contaminated environments.
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•Cr(VI) impact in sunflower roots was studied by LC-MS-based metabolomic approach.•Statistical PLS method was used to find compounds mostly affected by Cr(VI).•Eighteen compounds were annotated as linoleic, linoleic acids and related oxylipins.•Direct involvement of Cr(VI) in non-enzymatic oxidation of fatty acids is proposed.•Oxylipins as signaling molecules participate in sunflower defense against Cr(VI).
In this work,
Lactobacillus casei rhamnosus were obtained from the commercial product of fermented milk and possible antagonistic effect of selenium (as sodium selenite) against cadmium toxicity was ...studied. The bacteria capability to incorporate Se was demonstrated: after 1 week exposure to Se(IV), its total concentration in the freeze-dried biomass was 405
±
28
μg/g (7.4
±
0.8
μg/g in control). In the presence of Se(IV) and Cd(II), the bacterial growth and cell viability were improved and lipid peroxidation less marked with respect to bacteria exposed to Cd(II) alone. The distribution of Se and Cd in molecular mass fractions of bacteria extracts was investigated by size exclusion chromatography with diode array and ICP-MS detection. The results obtained suggest that the antagonistic effect of Se is due to lower incorporation of cadmium at a high molecular mass (MM
<
600
kDa). Slightly different distribution of elements in the fractions of MM
<
40
kDa suggests the formation of new chemical species involving Cd and Se in bacteria exposed to Cd(II)
+
Se(IV) as compared to those exposed to Cd(II) alone. The study illustrates the high utility of atomic spectrometry to critically inform molecular questions that could be important in the industrial processes based on bacterial activity.
Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated ...with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.
Mucor circinelloides
is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine ...the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of
adh
mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh
+
strain R7B, the ADH-defective (Adh
-
) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD
+
-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the
adhl
gene. Complementation with the wild-type allele
adh1
+
by transformation of M5 remedied all the defects caused by the
adh1
mutation. These findings indicate that in
M. circinelloides
, the product of the
adh1
gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of
M. circinelloides
possess low mRNA levels of the ethanol assimilation genes (
adl2
and
acs2
), which could explain their inability to grow in the alcohol.
Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular ...reduction of hexavalent chromium Cr(VI) to trivalent chromium Cr(III) may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system.
The purpose of this work was to gain an insight on the potential role of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici in the translocation of metals and metalloids from soil to ...plant roots in tomato (Lycopersicum esculentum). Two varieties of tomato (one susceptible and another resistant to infection by Fusarium oxysporum f. sp. lycopersici) were challenged with the fungus for different periods of time, and several elements (V, Cr, Mn, Co, Cu, Zn, As, Se, Mo, Ag, Cd, Pb) were determined in roots and in soil substrate. Additionally, phenolic plant products were also analyzed for the evaluation of the plant response to biotic stress. In order to obtain representative results for plants cultivated in noncontaminated environments, the infected and control plants were grown in commercial soil with natural, relatively low metal concentrations, partly associated with humic substances. Using such an experimental design, a specific role of the fungus could be observed, while possible effects of plant exposure to elevated concentrations of heavy metals were avoided. In the infected plants of two varieties, the root concentrations of several metals/metalloids were increased compared to control plants; however, the results obtained for elements and for phenolic compounds were significantly different in the two plant varieties. It is proposed that both Lycopersicum esculentum colonization by Fusarium oxysporum f. sp. lycopersici and the increase of metal bioavailability due to fungus-assisted solubilization of soil humic substances contribute to element traffic from soil to roots in tomato plant.
Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great ...interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg
−1
) and iron (from 2 to 108 g kg
−1
). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to
Bacillus
and
Lysobacter
genera.
Lysobacter
related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to
Thiobacillus
were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with
Bacillus
(already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest.
The application of capHPLC-ESI-QTOF-MS and MS/MS to study the impact of Cr(VI) on metabolites profile in Helianthus annuus is reported. Germinated seeds were grown hydroponically in the presence of ...Cr(VI) (25 mgCr/L) and root extracts of the exposed and control plants were analyzed by untargeted metabolomic approach. The main goal was to detect which metabolite groups were mostly affected by Cr(VI) stress; two data analysis tools (ProfileAnalysis, Bruker, and online XCMS) were used under criteria of intensity threshold 5 · 104 cps, fold change ≥ 5, and p≤0.01, yielding precursor ions. Molecular formulas were assigned based on data processing with two computational tools (SIRIUS and MS-Finder); annotation of candidate structures was performed by database search using CSI:FingerID and MS-Finder. Even though ultimate identification has not been achieved, it was demonstrated that secondary metabolism became activated under Cr(VI) stress. Among 42 candidate compounds returned from database search for seven molecular formulas, ten structures corresponded to isocoumarin derivatives and eleven were sesquiterpenes or sesquiterpene lactones; three benzofurans and four glycoside or pyrane derivatives of phenolic compounds were also suggested. To gain further insight on the effect of Cr(VI) in sunflower, isocoumarins and sesquiterpenes were selected as the target compounds for future study.
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