Chitinase gene sequences retrieved from diverse aquatic habitats reveal environment-specific distributions

Abstract

Chitin is an abundant biopolymer whose degradation is mediated primarily by bacterial chitinases. We developed a degenerate PCR primer set to amplify a approximately 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 10 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. We were unable to obtain PCR product with these primers from an alkaline, hypersaline lake (Mono Lake, California). 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 we found significant differences between libraries from different water column samples and between water column and sediment samples. However, identical sequences were retrieved from samples collected at widely distributed locations that did not necessarily represent similar environments, suggesting homogeneity of chitinoclastic communities between some environments.

FIG. 1.

Design of degenerate primers for family 18, group I chitinase genes. Alignments of chitinase amino acid sequences from organisms representing diverse phylogenetic lineages were used to design the degenerate primers. Symbols represent bacterial taxonomic groups: γ, γ-proteobacteria; β, β-proteobacteria; α, α-proteobacteria; and +, gram-positive bacteria. GenBank accession numbers are provided in parentheses. Position designations relative to the Serratia marcescens chitinase sequence (P07254) are shown above the alignment. Conserved residues are shown in black, similar residues in grey. I, inosine base; Y, C or T; W, A or T; S, G or C; and R, A or G. The degeneracy for both primers in this study is 16-fold. The references for chiAfor.ext and chiA.rev are this study and reference , respectively.

FIG. 2.

Neighbor-joining tree (partial sequence, ∼800 bp) showing phylogenetic relationships between family 18, group I chitinase nucleotide sequences. Clone designations are as follows: AOW55, Arctic Ocean, 55-m depth; AOW131, Arctic Ocean, 131-m depth; BBW, Bodega Bay water column; SIS, Sapelo Island sediments; SIW, Sapelo Island water column; SFBS, San Francisco Bay sediments; SFBW, San Francisco Bay water column; SJRW, San Joaquin River water column; SLW21, Soap Lake, 21-m depth; SLW23, Soap Lake, 23-m depth; TBS, Tomales Bay sediments; TLS, Topaz Lake sediments; and WLS, Walker Lake sediments. Water column samples for which no depth is given were collected at the surface (nominal depth, 0.1 m). Each sequence from a given library is also provided with a numerical designation. Branches containing identical sequences are indicated with a filled circle. The scale bar indicates Jukes-Cantor distance. Bootstrap values of >50% (for 100 iterations) are shown at branch nodes. The tree is unrooted with the chitinase gene from Bacillus circulans (AF154827) as the outgroup. GenBank accession numbers for reference sequences are provided in parentheses.

FIG. 3.

Conserved residues including and surrounding the catalytic domain of proteobacterial chitinases. Residues are coded according to degree of conservation as follows: black, >75%; gray, 50 to 75%; and no color, <50%. Positions that are altered in chitinase sequences retrieved in this study are indicated by symbols. Stars indicate residues found in a limited number of sequences and that are described in the text. Residues found exclusively in sequences retrieved from Soap Lake samples are circled. Numbers and dots indicate residue positions relative to the Serratia marcescens gene chiA (P07254).