Applied and Environmental Microbiology
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Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge ABSTRACT The environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5-seco pathway. PCR-based functional assays detected sequences closely related to alphaproteobacterialoecC , a key gene of the 4,5-seco pathway. Metagenomic analysis suggested thatNovosphingobium spp. are major estrogen degraders in estrone-amended activated sludge.Novosphingobium sp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 μg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance ofNovosphingobium spp. in activated sludge. IMPORTANCE Estrogens, which persistently contaminate surface water worldwide, have been classified as endocrine disruptors and human carcinogens. We contribute new knowledge on the major estrogen biodegradation pathway and estrogen degraders in wastewater treatment plants. This study considerably advances the understanding of environmental estrogen biodegradation, which is instrumental for the efficient elimination of these hazardous pollutants. Moreover, this study substantially improves the understanding of microbial estrogen degradation in the environment.
Applied and Environmental Microbiology - Tập 84 Số 10 - 2018
Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp ABSTRACT
Pseudomonas
sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of
mcbA
, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of
Escherichia coli
. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of
E. coli
expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent
K
m
and
V
max
in the range of 98 to 111 μM and 69 to 73 μmol · min
−1
· mg
−1
, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of
Pseudomonas
spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.
IMPORTANCE
Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of
Pseudomonas
spp. Predicted transmembrane domain and signal peptide sequences of
Pseudomonas
were found to be functional in
Escherichia coli
and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.
Applied and Environmental Microbiology - Tập 84 Số 2 - 2018
Preferential Utilization of Aromatic Compounds over Glucose by <i>Pseudomonas putida</i> CSV86 ABSTRACT
Pseudomonas putida
CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.
Applied and Environmental Microbiology - Tập 72 Số 3 - Trang 2226-2230 - 2006
Metabolism of Carbaryl via 1,2-Dihydroxynaphthalene by Soil Isolates <i>Pseudomonas</i> sp. Strains C4, C5, and C6 ABSTRACT
Pseudomonas
sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O
2
uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl → 1-naphthol → 1,2-dihydroxynaphthalene → salicylaldehyde → salicylate → gentisate → maleylpyruvate.
Applied and Environmental Microbiology - Tập 71 Số 10 - Trang 5951-5956 - 2005
Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in <i>Pseudomonas</i> sp. Strain C5pp
The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family.
Pseudomonas
sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.
Applied and Environmental Microbiology - Tập 84 Số 24 - 2018
Synergistic Actions of Nisin, Sublethal Ultrahigh Pressure, and Reduced Temperature on Bacteria and Yeast ABSTRACT
Nisin in combination with ultrahigh-pressure treatment (UHP) showed strong synergistic effects against
Lactobacillus plantarum
and
Escherichia coli
at reduced temperatures (<15°C). The strongest inactivation effects were observed when nisin was present during pressure treatment and in the recovery medium. Elimination (>6-log reductions) of
L. plantarum
was achieved at 10°C with synergistic combinations of 0.5 μg of nisin per ml at 150 MPa and 0.1 μg of nisin per ml at 200 MPa for 10 min. Additive effects of nisin and UHP accounted for only 1.2- and 3.7-log reductions, respectively. Elimination was also achieved for
E. coli
at 10°C with nisin present at 2 μg/ml, and 10 min of pressure at 200 MPa, whereas the additive effect accounted for only 2.6-log reductions. Slight effects were observed even against the yeast
Saccharomyces cerevisiae
with nisin present at 5 μg/ml and with 200 MPa of pressure. Combining nisin, UHP, and lowered temperature may allow considerable reduction in time and/or pressure of UHP treatments. Kill can be complete without the frequently encountered survival tails in UHP processing. The slightly enhanced synergistic kill with UHP at reduced temperatures was also observed for other antimicrobials, the synthetic peptides MB21 and histatin 5. The postulated mode of action was that the reduced temperature and the binding of peptides to the membrane increased the efficacy of UHP treatment. The increases in fatty acid saturation or diphosphatidylglycerol content and the lysylphosphatidyl content of the cytoplasm membrane of
L. plantarum
were correlated with increased susceptibility to UHP and nisin, respectively.
Applied and Environmental Microbiology - Tập 65 Số 9 - Trang 4148-4154 - 1999
Inhibitory effects of raw carrots on Listeria monocytogenes The survival and growth of two strains of Listeria monocytogenes on raw and cooked carrots stored at 5 and 15 degrees C and in carrot juice media at 30 degrees C were investigated. The influence of shredding, chlorine treatment, and packaging under an atmosphere containing 3% O2 and 97% N2 on the behavior of L. monocytogenes and naturally occurring microflora was determined. Populations of viable L. monocytogenes decreased upon contact with whole and shredded raw carrots but not cooked carrots. Viable populations also decreased in cell suspensions in which raw carrots were dipped. Small populations of L. monocytogenes detected on whole carrots immediately after dipping were essentially nondetectable after 7 days of storage at 5 or 15 degrees C. After a lag of 7 days at 5 degrees C, significant (P less than or equal to 0.05) increases in populations were detected on shredded carrots after 24 days of storage. Carrots stored at 5 or 15 degrees C spoiled before L. monocytogenes grew. Populations of mesophilic aerobes, psychrophiles, and yeasts and molds increased throughout storage at 5 and 15 degrees C. Cutting treatment (whole or shredded carrots), chlorine treatment, and modified-atmosphere packaging had no effect on the survival or growth of L. monocytogenes or naturally occurring microflora. The presence of raw carrot juice in tryptic phosphate broth at a concentration as low as 1% substantially reduced the maximum population of L. monocytogenes reached after 24 h at 30 degrees C. The anti-Listeria effect of carrots was essentially eliminated when the carrots were cooked.(ABSTRACT TRUNCATED AT 250 WORDS)
Applied and Environmental Microbiology - Tập 56 Số 6 - Trang 1734-1742 - 1990
Relationship between Membrane Damage and Cell Death in Pressure-Treated <i>Escherichia coli</i> Cells: Differences between Exponential- and Stationary-Phase Cells and Variation among Strains ABSTRACT
The relationship between membrane damage and loss of viability following pressure treatment was examined in
Escherichia coli
strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-β-
d
-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.
Applied and Environmental Microbiology - Tập 66 Số 7 - Trang 2829-2834 - 2000
Degradation of substituted indoles by an indole-degrading methanogenic consortium Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.
Applied and Environmental Microbiology - Tập 57 Số 9 - Trang 2622-2627 - 1991
Metabolism of 3-methylindole by a methanogenic consortium A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.
Applied and Environmental Microbiology - Tập 58 Số 8 - Trang 2667-2669 - 1992
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