IDENTIFICATION OF AGAROLYTIC BACTERIA PDF

  • IDENTIFICATION OF AGAROLYTIC BACTERIA PDF

    August 15, 2020 By admin 0 comments

    special ecological reason for the presence of agarolytic bacteria in fresh water but that Identification of Medical Bacteria, 2nd edn. Cam-. Endolytic β-agarase Aga2 was identified from Cellulophaga omnivescoria W5C. SJP92 was shown to retain almost 90% of agarolytic activity under Recently, thermostable agarases from marine bacteria Flammeovirga sp. Abstract: Agarolytic bacteria use agarase to utilize agar as sole source of carbon. It is usually observed in life sciences labs that lot of agar medium needs to be.

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    The phenotypic and agarolytic features if an unidentified marine bacteria that was isolated from the southern Pacific coast was investigated.

    The strain was gram negative, obligately aerobic, and polarly flagellated. On the basis of several phenotypic characters and a phylogenetic analysis of the genes coding for the 16S rRNA, this strain was identified as Pseudoalteromonas antarctica strain N In solid agar, this isolate produced a diffusible agarase that caused agar softening around the colonies. An extracellular agarase was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on DEAE-cellulose.

    The purified protein was determined to be homogeneous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it had a molecular mass of 33 kDa. Agar, a polysaccharide present in the cell walls of some red algae, can be degraded by several bacterial strains from marine environments and other sources.

    Some of the bacterial isolates have been assigned to the genera Alteromonas 12212733Cytophaga 43Streptomyces 36Vibrio 339and Pseudomonas Previous studies have shown that agar degradation identigication occur by two mechanisms that depend on the specificity of the cleaving enzymes.

    Finally, neoagarobiose is hydrolyzed to 3,6-anhydro- l -galactose and galactose in the cell cytoplasm by neoagarobiose hydrolase Agarotriose was the smallest product detected in this system. Further studies on the characterization of new agarases and their coding genes will be required to determine the significance of these identiflcation regions.

    In our laboratory, we have isolated a few agar-softening and agar-liquefying bacterial strains from the southern Chilean coast to characterize their extracellular agarases in an attempt to contribute to our understanding of the basis of agar hydrolysis. Previous results on the purification and characterization of an extracellular agarase from the agar-liquefying strain Alteromonas sp. We describe here the identification of a new identiffication bacterial strain, P.

    Strain N-1 was isolated from decomposing algae in Niebla Valdivia, Chile. The screening was carried out on agar plates in a medium containing 0. Colonies that formed pits or clearing zones on agar were picked up and purified further by the same plating fo.

    For liquid cultures, agar 0. Sugars were sterilized by filtration through 0. Guinea University of Barcelona, Barcelona, Spain Staining, morphology, and motility were determined as described by Cowan Oxidation and fermentation tests were done in MOF medium as recommended by Leifson 26but without agar. The type of flagellum was determined by negative staining with uranyl acetate and electron microscopy as described afarolytic Cole and Popkin Other biochemical and physiological tests were carried out essentially as described by Stolp and Gadkari 38 and Stanier et al.

    Genomic DNA was prepared by the procedure of Ausubel et al. The single DNA band of approximately 1. The sequence of the 16S rDNA of strain N-1 was aligned with the sequences of a number of Pseudoalteromonas strains available and was analyzed essentially as described by Gauthier et al. The GenBank accession number for the small subunit identificatiion P. An overnight culture of isolated colonies was prepared in a medium of the bqcteria composition as that of medium A, except that the agar concentration was lowered to 0.

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    Phenylmethylsulfonylfluoride PMSF was added to a final concentration of 0. Agarase activity was determined by the method of Dygert et al.

    An overnight culture of isolated colonies of strain N-1 was prepared in the medium described above and used to inoculate 2 liters of fresh medium containing 0.

    PMSF was added to a final concentration identificatin 0. The dialyzate was loaded onto a DEAE-cellulose agarolytiv 10 by 1. The protein was eluted batchwise with 90 ml of 1. Fractions 3 ml were collected, pooled on the basis of activity, and then loaded onto the DEAE-cellulose column 10 by 1. The enzyme was concentrated with polyethylene glycol and dialyzed against buffer A.

    The amount of protein in the column fractions was determined by measuring the A The amounts of protein in the pooled fractions were estimated by the method of Bradford 12 with fructose-1,6-bisphosphatase as the standard.

    Proteins were stained with Coomassie brilliant blue Agarolytif To characterize the hydrolysis products of agar with the purified enzyme, a solution of 0. The oligosaccharides were detected by evaluation of the refractive index. The undigested polysaccharides from the previous digest At identifkcation incubation times, the colonies produced a red-brown diffusible identificatin. The amount of pigment was dependent on the addition of tyrosine to the culture medium, suggesting the presence of a melaninlike pigment 2.

    Strain N-1 is a gram-negative rod bacterium, motile by a polar flagellum; it is also obligate aerobic, catalase and oxidase positive, and urease, indole, and arginine dihydrolase negative.

    It requires sodium ion icentification growth, has an oxidative metabolism, and does not accumulate polyhydroxybutyrate as an intracellular reserve.

    Based on this property, strain N-1 could be assigned to the vacteria Alteromonas 671820 or Pseudoalteromonas Strain N-1 can be distinguished from P. Strain N-1 can also be distinguished from S. In addition, strain N-1, unlike the Shewanella spp. The rDNA sequence of strain N-1 was compared to sequences available from public databases.

    Strain N-1 and P. Based in these data we propose agarolytkc assignment of our strain as P. However, we must point out that strain N-1 differs from the type strain of this species in some properties. The unrooted tree was constructed by neighbor-joining analysis.

    Percentages are indicated by bootstraps replicates for neighbor-joining analysis; replicates for parsimony. The highest level of agarase was reached during the stationary phase.

    At longer incubation periods the level of agarase decreases, a trend probably due to the presence of proteases.

    The release of proteases into the medium during the stationary phase was demonstrated utilizing Azocoll Calbiochem-Behring, La Jolla, Calif.

    In the presence of agar, glucose or galactose did not affect the production of agarase in this strain data not shown.

    No activity was observed when other carbon sources, such as glucose or galactose, were used instead of identificatiion as the sole carbon source.

    Strain N-1 was cultured in liquid medium containing 0. Purification was attempted after 30 h of incubation. The enzyme was purified by taking advantage of its high binding affinity to DEAE-cellulose when loaded at low salt concentrations at cruder stages. The enzyme was slowly released from the DEAE-cellulose by a washing with 1.

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    Additional purification of the enzyme was achieved by gel filtration on Sephadex G75 Fig. At this identificarion the enzyme eluted in the flowthrough, indicating that the strong binding seen at the beginning of the purification could be mediated by an unidentified extracellular component of this strain or by an agar-derived product that is separated during the gel filtration step.

    Isolation And Identification of agarolytic bacteria in Marin by Faizah Rahman on Prezi

    The enzyme gave a single band on SDS-polyacrylamide gels Fig. Lane 1, molecular mass standards; lane 2, purified agarase ca. Agarase N-1 had a molecular mass of 33 kDa, as determined by a comparison with the mobility of protein standards Fig. The molecular mass of the enzyme was estimated by gel filtration by using Sephadex G25 and Superdex 75 columns.

    In both cases the enzyme showed a molecular mass of 16 kDa, indicating an interaction with these resins. The pH profile of agarase from strain N-1 was bell shaped, with a maximum at pH 7. The enzyme was stable under the conditions of this assay as determined by measuring the residual activity at pH 7.

    A Effect of pH on the activity of the purified agarase. The activity was determined at a pH between 3.

    B Effect of temperature on the stability of agarase from P. The enzyme was incubated in 50 mM sodium phosphate at pH 6. In contrast to the agarases from P.

    When enzyme activity was measured in the presence of NaCl in concentrations of up to 0. The assays were carried out in 50 mM phosphate pH 7.

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    K m values of 0. As shown by the HPLC profile, the purified enzyme from strain N-1 hydrolyzed agar to give two main oligosaccharide products Fig.

    The identities of these oligosaccharides were confirmed by thin-layer chromatographic analysis on silica gel plates not shown. There was no evidence of a signal at Hydrolysis products of agar by agarase from P.

    The oligosaccharides were detected by determining the refractive index with a detector Gilson, Middleton, Wis. Identificatiom Oligosaccharides released by agarase.

    Agar clearing, softening, and depressions around the colonies is characteristic for bacteria in groups 1 and 2.

    This effect would be related to the production of ieentification agarases that can diffuse though the gel pores. Cleavage of the polysaccharide chains causes agar softening and allows faster evaporation of water, leading to the formation of depressions.

    The exception is Alteromonas sp. We describe here the characterization of a new agarolytic bacterium isolated from the southern Chilean coast. This strain was identified as P. An extracellular agarase was purified to homogeneity in high yield by gel filtration and two steps of ion-exchange chromatography on DEAE-cellulose.

    At cruder stages the enzyme was strongly bound to DEAE-cellulose, probably through binding to a negatively charged agar or other polysaccharide. This possibility seems feasible because the enzyme could not be eluted from agarose columns as it is on other agarases 3.

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