caa a22 4500 60549875X CHVBK 20210128100548.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6639-5 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6639-5 A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil [Elektronische Daten] [Mariana Cretoiu, Francesca Berini, Anna Kielak, Flavia Marinelli, Jan van Elsas] 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. The Author(s), 2015 Chitinolytic enzymes nationallicence Fosmid library nationallicence Functional metagenomics nationallicence Suppressive soil nationallicence Cretoiu Mariana Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands aut Berini Francesca Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy aut Kielak Anna Department of Microbial Ecology, The Netherlands Institute of Ecology (NIOO), Wageningen, The Netherlands aut Marinelli Flavia Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy aut van Elsas Jan Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 8199-8215 0175-7598 99:19<8199 2015 99 253 https://doi.org/10.1007/s00253-015-6639-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6639-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Cretoiu Mariana Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands aut NATIONALLICENCE 700 1- Berini Francesca Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy aut NATIONALLICENCE 700 1- Kielak Anna Department of Microbial Ecology, The Netherlands Institute of Ecology (NIOO), Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Marinelli Flavia Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy aut NATIONALLICENCE 700 1- van Elsas Jan Department of Microbial Ecology, CEES, University of Groningen, Groningen, The Netherlands aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 8199-8215 0175-7598 99:19<8199 2015 99 253