Molecular analysis of carbon monoxide-oxidizing bacteria associated with recent Hawaiian volcanic deposits

Abstract

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.

FIG. 1.

Neighbor-joining analysis (1,000 bootstrap replicates) of inferred amino acid sequences for nonredundant coxL clone sequences and known CO-oxidizing isolates. A total of 109 clone sequences (163 amino acid residues each) from the Forest (FOR), Pu'u Puai (PP), Halema'uma'u (HM), and the Caldera Rim (CR) libraries were analyzed. OMP and BMS clades are indicated based on known coxL gene sequences (13). Phylogenetic lineages are designated based on affiliations with known CO oxidizers. Bootstrap values of <70% are not shown. The Pyrobaculum aerophilum putative coxL sequence was used as an outgroup. The GenBank accession numbers for the following strains are given, with the clade indicated in parentheses. Aminobacter strain (str.) COX, AY307908 (BMS); Bradyrhizobium japonicum USDA 6, AY307921 (BMS); Bradyrhizobium strain CPP, AY307900 (BMS) and AY307913 (OMP); Burkholderia fungorum LB400, AY307901 (BMS), AY307916 (OMP); Burkholderia strain LUP, AY307907 (BMS); Mesorhizobium strain NMB1, AY307906 (BMS); Mycobacterium smegmatis, AY307916 (OMP); Oligotropha carboxydovorans, X82447(OMP); Pseudomonas thermocarboxydovorans, Y77931 (OMP); Stappia aggregata, AY307904 (BMS) and AY307918 (OMP); Stappia strain KB812, AY307898 (BMS) and AY307193 (OMP); Stappia strain KB902, AY307899 (BMS), AY307914 (OMP); Stappia strain M4, AY307902 (BMS) and AY307916 (OMP); Stappia strain M8, AY307903 (BMS) and AY307917 (OMP); Stappia stellulata, AY307905 (BMS) and AY307919 (OMP); Stenotrophomonas strain LUP, AY307920 (OMP); and Xanthobacter strain COX, AY307911 (BMS).

FIG. 2.

Frequency distribution of pairwise mismatches within coxL clone libraries. (A) Caldera Rim (•) and Halema'uma'u (○); (B) Forest (•) and Pu'u Puai (○).

FIG. 3.

Cumulative distribution of mismatch as a function of the number of mismatches. Forest, •; Caldera Rim, ▪; Halema'uma'u, □; Pu'u Puai, ○.

FIG. 4.

Correlation of microbial biomass (r = 0.99; ○) and respiration (r = 0.99; •) with average pairwise difference within clone libraries (Table 1). Biomass and respiration data from King (11). gdw, grams (dry weight).