Chitinolytic bacteria and enzymes from Mono Lake, CA, USA

• Main Author: LeCleir, Gary Raymond
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: uga 2005
• Subjects: Mono Lake; chitin; chitinolytic enzymes; n-acetylglucosaminidase; Bacteria; Vibrio; diversity; biogeography; Arctic; Arctic Ocean
• Online Access: http://hdl.handle.net/10724/22950; http://purl.galileo.usg.edu/uga_etd/lecleir_gary_r_200512_phd

Chitin is an abundant biopolymer whose degradation is mediated primarily by bacterial chitinases. We developed a degenerate PCR primer set to amplify a ~900 bp fragment of family 18, group I chitinase genes and used it to retrieve these gene fragments from environmental samples. Clone libraries of presumptive chitinase genes were created for nine water and six sediment samples from ten aquatic environments including freshwater and saline lakes, estuarine water and sediments and the central Arctic Ocean. Putative chitinase sequences were also retrieved from the Sargasso Sea metagenome sequence database. PCR product using these primers was not obtained from an alkaline, hypersaline lake (Mono Lake, CA). In total, 108 partial chitinase gene sequences were analyzed, with a minimum of 5 and a maximum of 13 chitinase sequences obtained from each library. All chitinase sequences were novel compared to previously identified sequences. Intralibrary sequence diversity was low, while significant differences were found between libraries from different water column samples and between water column and sediment samples. Identical sequences, however, were retrieved from samples collected at widely distributed locations that did not necessarily represent similar environments, suggesting homogeneity of chitinoclastic communities between some environments. An inability to amplify chitinase genes from Mono Lake, despite high levels of chitinolytic activity, prompted the analysis of the microbial community composition associated with Artemia monica exuvia and in chitin enrichments of Mono Lake water. Chitinolytic bacteria from Mono Lake were also isolated. Bacterial assemblages were characterized by cloning and sequencing 16S rDNA amplicons. Isolates were screened for chitinolytic activity using methylumbelliferyl-diacetylchitobioside (MUF-DC) and methylumbelliferyltriacetylchitotrioside (MUF-TC); for the ability to hydrolyze colloidal chitin; and for growth on medium containing only chitin. Several ribotypes were common to Artemia exuvia samples and chitin enrichments. Four Proteobacteria ribotypes were only retrieved from clone libraries of chitin enrichments. The majority of the isolates obtained were Gram-positive bacteria and 70% of the Gram-positive isolates hydrolyzed at least one model substrate. Chitinolytic genes from two Mono Lake isolates and from an environmental DNA library from Sapelo Island, GA were then obtained by shotgun cloning using fosmid vectors. Fosmid libraries were screened for MUF-DC hydrolysis and six positive clones were analyzed further. Genes of interest were localized by random transposon mutagenesis. One clone from a Mono Lake isolate contains a gene encoding a family 18 glycosyl hydrolase. Two additional clones, one from a Mono Lake isolate and another from the environmental library, contain genes encoding family 20 glycosyl hydrolases. The proteins expressed by these clones were characterized with respect to pH and salt tolerance. An enzyme from Mono Lake clone AI214B1 maintained activity at pH 11 and salinity of 225 ppt. These characteristics have not been previously associated with this enzyme family. PhD Marine Sciences Marine Sciences James T. Hollibaugh James T. Hollibaugh Samantha Joye Mary Ann Moran Brian Binder Patricia Yager