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1.
Arch Microbiol ; 206(6): 252, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38727820

RESUMEN

A microaerophilic Gram-stain-negative bacilliform bacterial strain, FB-5 T, was isolated from activated sludge in Yokohama, Japan, that exhibited filamentous growth and formed a microtube (sheath). Cells were motile using a single polar flagellum. The optimum growth temperature and pH were 30 °C and 7.5, respectively. Strain FB-5 T was catalase-negative. Peptides and amino acids were utilized as energy and carbon sources. Sugars and organic acids were not utilized. Vitamin B12 enhanced the growth of strain FB-5 T. Sulfur-dependent lithotrophic growth was possible. Major respiratory quinone was UQ-8. Major fatty acids were C16:1ω7 and C16:0. The genomic DNA G + C content was 69.16%. Phylogenetic analysis of the 16S rRNA gene suggested that strain FB-5 T belongs to the genus Sphaerotilus. The close relatives were S. natans subsup. sulfidivorans and S. natans subsup. natans with 98.0% and 97.8% similarity based on the 16S rRNA gene analysis, respectively. The genome size (6.06 Mbp) was larger than that (4.39-5.07 Mbp) of the Sphaerotilus strains. The AAI values against the related strains ranged from 71.0 to 72.5%. The range of ANI values was 81.7 - 82.5%. In addition to these distinguishable features of the genome, the core genome and dDDH analyses suggested that this strain is a novel member of the genus Sphaerotilus. Based on its physiological properties and genomic features, strain FB-5 T is considered as a novel species of the genus Sphaerotilus, for which the name S. microaerophilus sp. nov. is proposed. The type strain is FB-5 T (= JCM 35424 T = KACC 23146 T).


Asunto(s)
Composición de Base , ADN Bacteriano , Ácidos Grasos , Filogenia , ARN Ribosómico 16S , Aguas del Alcantarillado , Aguas del Alcantarillado/microbiología , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Análisis de Secuencia de ADN , Japón , Genoma Bacteriano
2.
Appl Environ Microbiol ; 89(4): e0191922, 2023 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-36951572

RESUMEN

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.


Asunto(s)
Glicosiltransferasas , Leptothrix , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Leptothrix/fisiología , Calcio/metabolismo , Análisis de Secuencia de ADN , Glicoconjugados/metabolismo
3.
J Appl Microbiol ; 134(9)2023 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-37660239

RESUMEN

AIMS: Enzymatic degradation of ß-1,4-linked glucose and glucosamine (glucosaminoglucan, GG), which is prepared from Thiothrix nivea and can act as a cellulose-aminating agent with a strong affinity to cellulose, was attempted. METHODS AND RESULTS: A chitosanase-secreting fungal strain was isolated as a GG-degrading microbe. GG was found to be degraded by not only chitosanases but also cellulases. Based on nuclear magnetic resonance spectroscopy, both enzymes were found to produce GlcN-Glc from GG. The cellulases also produced GlcN-Glc-GlcN-Glc as an additional final digest. Furthermore, aminated (GG-coated) cellulose nanofibers exhibited cellulase resistance. The flexibility of GG adsorbed onto a cellulose crystal was almost identical to that of cellulose, as estimated via the molecular dynamics calculations. CONCLUSIONS: The chitosanase and cellulase hydrolyzed the ß-1,4-linkage from Glc to GlcN and were expected to recognize the tetramer and hexamer units of GG depending on their final products. The cellulose nanofibers acquired cellulase resistance via amination with GG, probably because of the lower activity of cellulase to GG than cellulose.


Asunto(s)
Celulasa , Nanofibras , Transporte Biológico , Celulosa , Glucosa
4.
Biosci Biotechnol Biochem ; 87(3): 256-266, 2023 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-36535637

RESUMEN

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-ß-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.


Asunto(s)
Ácido Hialurónico , Aguas del Alcantarillado , Ácido Hialurónico/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Bacteroidetes , Glicosaminoglicanos , Bacterias/metabolismo
5.
Appl Environ Microbiol ; 88(23): e0134122, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36416549

RESUMEN

The bacterium Leptothrix cholodnii generates filaments encased in a sheath comprised of woven nanofibrils. In static liquid culture, L. cholodnii moves toward the air-liquid interface, where it forms porous pellicles. Observations of aggregation at the interface reveal that clusters consisting of only a few bacteria primarily grow by netting free cells. These growing clusters hierarchically enlarge through the random docking of other small clusters. We find that the bacteria swim using their polar flagellum toward the interface, where their sheath assists them in intertwining with others and thereby promotes the formation of small clusters. In contrast, sheathless hydrophobic mutant cells get stuck to the interface. We find that the nanofibril sheath is vital for robust pellicle formation as it lowers cell surface hydrophobicity by 60%, thereby reducing their adsorption and enabling cells to move toward and stick together at the air-liquid interface. IMPORTANCE Efficient and sustainable management of water resources is becoming a fundamental issue for supporting growing populations and for developing economic activity. Fundamental to this management is the treatment of wastewater. Microorganisms are the active component of activated sludge that is employed in the biodegradation process of many wastewater treatment facilities. However, uncontrolled growth of filamentous bacteria such as Sphaerotilus often results in filamentous bulking, lowering the efficiency of water treatment systems. To prevent this undesirable condition, strategies based on a fundamental understanding of the ecology of filamentous bacteria are required. Although the filamentous bacterium Leptothrix cholodnii, which is closely related to Sphaerotilus, is a minor inhabitant of activated sludge, its complete genome sequence is known, making gene manipulation relatively easy. Moreover, L. cholodnii generates porous pellicles under static conditions, which may be a characteristic of filamentous bulking. We show that both swimming motility and nanofibril-mediated air-liquid interface attachment are required for porous pellicle formation. These insights are critical for a better understanding of the characteristics of filamentous bulking and might improve strategies to control activated sludge.


Asunto(s)
Leptothrix , Aguas del Alcantarillado/microbiología , Porosidad , Aguas Residuales , Bacterias/metabolismo
6.
J Appl Microbiol ; 133(2): 607-618, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-35462453

RESUMEN

AIMS: This study aimed to unveil perrhenate sorption properties of the filamentous sheaths formed by Sphaerotilus montanus, Sphaerotilus natans and Thiothrix fructosivorans. METHODS AND RESULTS: The adsorptions of perrhenate on lyophilizates of the above-mentioned filamentous sheaths were analysed by ICP, IR, XPS and EDX. The capacity reached 82 mg per g-adsorbent, when using S. natans. The Langmuir coefficient of this adsorbent was found to be the largest of the three. The adsorption capacity was discussed with respect to the amount of nitrogen and phosphorus in the adsorbents. The occurrence of anion exchange was implied by the IR spectrum changes before and after adsorption. The adsorption data fitted well with a pseudo-second-order equation, suggesting that the rate is determined by the chemical bond formation. CONCLUSIONS: A significant amount of perrhenate was adsorbed on the sheaths formed by S. montanus, S. natans and T. fructosivorans. The adsorption was correlated with the elemental compositions. A strong chemical bond formation was suggested from the results of the Langmuir adsorption isotherm and kinetic analysis. SIGNIFICANCE AND IMPACT OF STUDY: The capacity obtained for S. natans is one of the largest adsorptions amongst the similar biomaterials, implying the possibility of providing economical adsorbents of rare metal oxyanions.


Asunto(s)
Sphaerotilus , Adsorción , Iones , Cinética , Renio , Thiothrix
7.
Biosci Biotechnol Biochem ; 84(10): 2085-2095, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32594856

RESUMEN

ß-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates.


Asunto(s)
Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Azufre/metabolismo , Thiothrix/metabolismo , Peso Molecular , Oxidación-Reducción , Solubilidad
8.
Arch Microbiol ; 200(8): 1257-1265, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29934786

RESUMEN

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.


Asunto(s)
Thiothrix/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Thiothrix/química , Thiothrix/genética
9.
Sensors (Basel) ; 18(11)2018 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-30380790

RESUMEN

Recently, a project was initiated in Japan to transport a large amount of liquid hydrogen (LH2) from Australia to Japan by sea. It is important to understand the sloshing and boil-off that are likely to occur inside an LH2 tank during marine transportation by ship, but such characteristics are yet to be experimentally clarified. To do so, we combined the liquid level detected by five 500 mm long external-heating-type magnesium diboride (MgB2) level sensors with synchronous measurements of temperature, pressure, ship motion, and acceleration during a zigzag maneuver. During this zigzag maneuver, the pressure of gaseous hydrogen (GH2) in the small LH2 tank increased to roughly 0.67 MPaG/h, and the temperature of the GH2 in the small LH2 tank increased at the position of gaseous hydrogen at roughly 1.0 K/min when the maximum rolling angle was 5°; the average rolling and liquid-oscillation periods were 114 and 118 s, respectively, as detected by the MgB2 level sensors, which therefore detected a long-period LH2 wave due to the ship's motion.

10.
Biosci Biotechnol Biochem ; 80(6): 1155-63, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26923741

RESUMEN

A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP(+)-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/metabolismo , Glicoles/metabolismo , Mycobacterium/enzimología , Ácidos Pentanoicos/metabolismo , Microbiología del Suelo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Expresión Génica , Glicoles/química , Concentración de Iones de Hidrógeno , Cinética , Mycobacterium/clasificación , Mycobacterium/genética , Sistemas de Lectura Abierta , Oxidación-Reducción , Pentanos/química , Pentanos/metabolismo , Ácidos Pentanoicos/química , Filogenia , Propilenglicol/química , Propilenglicol/metabolismo , Multimerización de Proteína , Alineación de Secuencia , Estereoisomerismo , Especificidad por Sustrato
11.
Biosci Biotechnol Biochem ; 78(11): 1879-86, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25082080

RESUMEN

An enzyme capable of reducing acetoin in the presence of NADH was purified from Mycobacterium sp. B-009, a non-clinical bacterial strain of soil origin. The enzyme is a homotetramer and can be classified as a medium-chain alcohol dehydrogenase/reductase based on the molecular weight of the monomer. Identification of the structural gene revealed a limited distribution of homologous genes only among actinomycetes. In addition to its activity as a reductase specific for (S)-acetoin (EC 1.1.1.76), the enzyme showed both diacetyl reductase (EC 1.1.1.304) and NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) activities. (S)-Acetoin and diacetyl reductases belong to a group of short-chain alcohol dehydrogenase/reductases but do not have superior abilities to dehydrogenate monoalcohols. Thus, the purified enzyme can be readily distinguished from other enzymes. We used the dual functionality of the enzyme to effectively reduce diacetyl to (S)-acetoin, coupled with the oxidation of 1-butanol.


Asunto(s)
Acetoína/metabolismo , Alcohol Deshidrogenasa/metabolismo , Diacetil/metabolismo , Mycobacterium/enzimología , Acetoína/química , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Secuencia de Aminoácidos , Diacetil/química , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Mycobacterium/clasificación , Oxidación-Reducción , Filogenia , Especificidad por Sustrato
12.
Bio Protoc ; 13(8): e4652, 2023 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-37113333

RESUMEN

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaLeptothrix cholodniiSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withEscherichia coliS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.

13.
Arch Microbiol ; 194(8): 667-73, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22392226

RESUMEN

Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class ß-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chemical perspective, the sheath can be described as a supermolecule composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, we attempted to detect sheath elongation through microscopic examination by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, we assumed that the newly secreted thiopeptidoglycan molecules are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidation in microcultures was also examined with respect to cultivation time. Results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.


Asunto(s)
Leptothrix/fisiología , Manganeso/química , Proliferación Celular , Glicoconjugados/metabolismo , Leptothrix/citología , Leptothrix/metabolismo , Leptothrix/ultraestructura , Microscopía Electrónica de Rastreo , Oxidación-Reducción
14.
Biosci Biotechnol Biochem ; 76(12): 2357-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23221712

RESUMEN

Sphaerotilus natans is a filamentous sheath-forming bacterium. A method of selective fluorescent-labeling of its sheath using conventional reagents was developed. Terminal expansion of the sheath was confirmed by this method. In addition, ubiquitous cell growth was revealed by sequential phase-contrast microscopy of a filament. Based on this and earlier reports, a model of the sheath formation is proposed.


Asunto(s)
Microtúbulos/metabolismo , Sphaerotilus/citología , Proliferación Celular , Modelos Biológicos
15.
ACS Omega ; 7(45): 41768-41774, 2022 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-36406563

RESUMEN

The resistive switching temperature associated with the metal-insulator transition (MIT) of epitaxial VO2 thin films grown on flexible synthetic mica was modulated by bending stress. The resistive switching temperature of polycrystalline VO2 and V2O5 thin films, initially grown on synthetic mica without a buffer layer, was observed not to shift with bending stress. By inserting a SnO2 buffer layer, epitaxial growth of the VO2 (010) thin film was achieved, and the MIT temperature was found to vary with the bending stress. Thus, it was revealed that the bending response of the VO2 thin film depends on the presence or absence of the SnO2 buffer layer. The bending stress applied a maximum in-plane tensile strain of 0.077%, resulting in a high-temperature shift of 2.3 °C during heating and 1.8 °C during cooling. After 104 bending cycles at a radius of curvature R = 10 mm, it was demonstrated that the epitaxial VO2 thin film exhibits resistive switching temperature associated with MIT.

16.
Biosci Biotechnol Biochem ; 75(12): 2384-9, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22146728

RESUMEN

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD(+)-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Mycobacterium/enzimología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/aislamiento & purificación , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mycobacterium/clasificación , Mycobacterium/genética , Fenotipo , Filogenia , Propilenglicol/química , Propilenglicol/metabolismo , Estereoisomerismo , Especificidad por Sustrato
17.
Int J Biol Macromol ; 183: 992-1001, 2021 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-33964269

RESUMEN

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.


Asunto(s)
Polisacárido Liasas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Sphaerotilus/química , Paenibacillus/enzimología
18.
PLoS One ; 15(1): e0227567, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31968006

RESUMEN

Prolific heterotrophic biofilm growth is a common occurrence in airport receiving streams containing deicers and anti-icers, which are composed of low-molecular weight organic compounds. This study investigated biofilm spatiotemporal patterns and responses to concurrent and antecedent (i.e., preceding biofilm sampling) environmental conditions at stream sites upstream and downstream from Milwaukee Mitchell International Airport in Milwaukee, Wisconsin, during two deicing seasons (2009-2010; 2010-2011). Biofilm abundance and community composition were investigated along spatial and temporal gradients using field surveys and microarray analyses, respectively. Given the recognized role of Sphaerotilus in organically enriched environments, additional analyses were pursued to specifically characterize its abundance: a consensus sthA sequence was determined via comparison of whole metagenome sequences with a previously identified sthA sequence, the primers developed for this gene were used to characterize relative Sphaerotilus abundance using quantitative real-time PCR, and a Sphaerotilus strain was isolated to validate the determined sthA sequence. Results indicated that biofilm abundance was stimulated by elevated antecedent chemical oxygen demand concentrations, a surrogate for deicer concentrations, with minimal biofilm volumes observed when antecedent chemical oxygen demand concentrations remained below 48 mg/L. Biofilms were composed of diverse communities (including sheathed bacterium Thiothrix) whose composition appeared to shift in relation to antecedent temperature and chemical oxygen demand. The relative abundance of sthA correlated most strongly with heterotrophic biofilm volume (positive) and dissolved oxygen (negative), indicating that Sphaerotilus was likely a consistent biofilm member and thrived under low oxygen conditions. Additional investigations identified the isolate as a new strain of Sphaerotilus montanus (strain KMKE) able to use deicer components as carbon sources and found that stream dissolved oxygen concentrations related inversely to biofilm volume as well as to antecedent temperature and chemical oxygen demand. The airport setting provides insight into potential consequences of widescale adoption of organic deicers for roadway deicing.


Asunto(s)
Biopelículas/efectos de los fármacos , Hielo , Compuestos Orgánicos/toxicidad , Ríos/química , Contaminantes Químicos del Agua/toxicidad , Biopelículas/crecimiento & desarrollo , Modelos Lineales , Metagenómica , Sphaerotilus/efectos de los fármacos , Sphaerotilus/genética , Sphaerotilus/fisiología
19.
Sci Rep ; 10(1): 6809, 2020 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-32321952

RESUMEN

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.


Asunto(s)
Chloroflexi/crecimiento & desarrollo , Purificación del Agua , Aerobiosis , Anaerobiosis , Chloroflexi/genética , Dosificación de Gen , ARN Ribosómico 16S/genética , Análisis de Regresión , Aguas del Alcantarillado/microbiología , Factores de Tiempo
20.
Int J Biol Macromol ; 109: 323-328, 2018 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-29253548

RESUMEN

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium commonly found in activated sludge. The filament of this bacterium is covered with a sheath. The sheath is an assemblage of macromolecular glucosaminoglucan (GG), [4)-ß-d-GlcN-(1 → 4)-ß-d-Glc-(1 → ]n, modified with an unidentified deoxy-sugar at position 3 of Glc. GG was obtained by dialysis after the partial hydrolysis of the sheath. The GG hydrogel was prepared by drying a GG solution. Then, the hydrogel was N-acetylated to prepare a stable hydrogel of N-acetylglucosaminoglucan (NGG), [4)-ß-d-GlcNAc-(1 → 4)-ß-d-Glc-(1 → ]n. The NGG hydrogel was stable in phosphate buffer but was disrupted by lysozyme addition, suggesting that NGG is susceptible to lysozyme degradation and has potential for medical use. The GG solution was N-acetylated to prepare a NGG suspension to confirm enzymatic degradation. The turbidity of the NGG suspension was decreased by lysozyme addition. Sugars released in the reaction mixture were derivatized with 4-aminobenzoic acid ethyl ester (ABEE) followed by HPLC analysis. Two major derivatives were detected, and their concentration was increased in reverse proportion to the turbidity of the reaction mixture. The derivatives were identified as GlcNAc-Glc-GlcNAc-Glc-ABEE and GlcNAc-Glc-ABEE by mass spectrometry. Consequently, NGG was found to be degraded by lysozyme via a mechanism similar to that of chitin degradation.


Asunto(s)
Acetilglucosamina/química , Biodegradación Ambiental , Glucanos/química , Glucanos/metabolismo , Thiothrix/química , Thiothrix/metabolismo , Cromatografía Líquida de Alta Presión , Hidrogeles , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier
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