A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil
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| 024 | 7 | 0 | |a 10.1007/s00253-015-6639-5 |2 doi |
| 035 | |a (NATIONALLICENCE)springer-10.1007/s00253-015-6639-5 | ||
| 245 | 0 | 2 | |a A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil |h [Elektronische Daten] |c [Mariana Cretoiu, Francesca Berini, Anna Kielak, Flavia Marinelli, Jan van Elsas] |
| 520 | 3 | |a Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40kb, yielding a total of approximately 5.8GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase. | |
| 540 | |a The Author(s), 2015 | ||
| 690 | 7 | |a Chitinolytic enzymes |2 nationallicence | |
| 690 | 7 | |a Fosmid library |2 nationallicence | |
| 690 | 7 | |a Functional metagenomics |2 nationallicence | |
| 690 | 7 | |a Suppressive soil |2 nationallicence | |
| 700 | 1 | |a Cretoiu |D Mariana |u Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands |4 aut | |
| 700 | 1 | |a Berini |D Francesca |u Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy |4 aut | |
| 700 | 1 | |a Kielak |D Anna |u Department of Microbial Ecology, The Netherlands Institute of Ecology (NIOO), Wageningen, The Netherlands |4 aut | |
| 700 | 1 | |a Marinelli |D Flavia |u Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy |4 aut | |
| 700 | 1 | |a van Elsas |D Jan |u Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/19(2015-10-01), 8199-8215 |x 0175-7598 |q 99:19<8199 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-015-6639-5 |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-015-6639-5 |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Cretoiu |D Mariana |u Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Berini |D Francesca |u Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Kielak |D Anna |u Department of Microbial Ecology, The Netherlands Institute of Ecology (NIOO), Wageningen, The Netherlands |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Marinelli |D Flavia |u Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a van Elsas |D Jan |u Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/19(2015-10-01), 8199-8215 |x 0175-7598 |q 99:19<8199 |1 2015 |2 99 |o 253 | ||