Succession and diel transcriptional response of the glycolate-utilizing component of the bacterial community during a spring phytoplankton bloom

Abstract

The influence of the phytoplankton-specific organic compound glycolate on bacterial community structure was examined during the 2004 spring phytoplankton bloom (February to April) in Dabob Bay in Washington. The diversity of the bacteria able to utilize glycolate during the phytoplankton bloom was determined using previously developed PCR primers to amplify the gene for the D subunit of glycolate oxidase (glcD). Many of the glcD sequences obtained represented novel sequences that appeared to be specific to marine environments. Overall, the glcD sequence diversity decreased as the phytoplankton bloom progressed. Phylotype-specific glcD quantitative PCR primers were designed for the six most commonly detected glcD phylotypes that represented distinct phylogenetic groups of heterotrophic bacteria. Three patterns of phylotype abundance were detected: four phylotypes were most abundant during the onset of the bloom; the abundance of one phylotype increased as the bloom progressed; and one phylotype was abundant throughout the bloom. Quantitative reverse transcriptase PCR with the same phylotype-specific primers was used to determine the levels of day and night glcD RNA transcription over the course of the bloom. glcD transcripts, when detectable, were always more abundant in the day than at night for each phylotype, suggesting that the bacteria responded to the glycolate produced by phytoplankton during the day. The nearly constant low in situ glycolate concentrations suggested that bacteria rapidly utilized the available glycolate. This study provided evidence for direct phytoplankton-bacterium interactions and the resulting succession in a single functional group of marine bacteria.

FIG. 1.

Rarefaction curves for the five glcD clone libraries constructed over the course of the bloom. An OTU was defined by using a level of DNA sequence similarity of 97%.

FIG. 2.

Maximum-likelihood phylogenetic trees for the GlcD amino acid sequences from Dabob Bay (DB) and from previously described samples collected in 2000 from Parks Bay in Washington (PB), San Juan Channel in Washington (SJC), and Chl a maximum (Chl) and surface (Surf) from an Atlantic Gulf Stream Ring. An initial tree containing all sequences supported (bootstrap value, 65) (not shown) the separation of clades for construction of two separate trees (A and B). The numbers in parentheses are the numbers of clones observed for the OTUs. For Dabob Bay, the five numbers in parentheses are in order for the five sequential clone libraries. Scale bars represent 1 substitution per 10 bases. OTUs for which phylotype-specific primers were designed for qPCR analyses are indicated by bold type. Methanosarcina barkeri strain fusaro was used as the outgroup for both trees. Unsupported branches (bootstrap values, <50) have been collapsed.

FIG. 2.

Maximum-likelihood phylogenetic trees for the GlcD amino acid sequences from Dabob Bay (DB) and from previously described samples collected in 2000 from Parks Bay in Washington (PB), San Juan Channel in Washington (SJC), and Chl a maximum (Chl) and surface (Surf) from an Atlantic Gulf Stream Ring. An initial tree containing all sequences supported (bootstrap value, 65) (not shown) the separation of clades for construction of two separate trees (A and B). The numbers in parentheses are the numbers of clones observed for the OTUs. For Dabob Bay, the five numbers in parentheses are in order for the five sequential clone libraries. Scale bars represent 1 substitution per 10 bases. OTUs for which phylotype-specific primers were designed for qPCR analyses are indicated by bold type. Methanosarcina barkeri strain fusaro was used as the outgroup for both trees. Unsupported branches (bootstrap values, <50) have been collapsed.

FIG. 3.

glcD copy number normalized to bacterial abundance (0.8- to 10-μm fraction) for one night and the following “noon” sample for each week of sampling. (A and B) Percentages of lower-abundance OTUs detected at night (A) and around noon (B) the next day. (C and D) Percentages of higher-abundance OTUs detected at night (C) and around noon (D) the next day. The OTU 68 values are the sums of the values for the two subgroups (OTU 68a and OTU 68b). The error bars indicate standard deviations for triplicate qPCR measurements of one sample per time. BD, below the limit of detection. Note that the y axis scales are different for different graphs.

FIG. 4.

glcD RNA transcript abundance normalized to the corresponding DNA abundance for OTU 52 (A), OTU 62 (B), OTU 68a (C), and OTU 68b (D). Data for OTU 68 are presented separately for the two subgroups amplified with different primer sets. The error bars indicate standard deviations for triplicate qPCR measurements of one sample per time for both DNA and RNA. BD, below the limit of detection. Note that the y axis scale for panel A differs from the y axis scale for panels B to D.

FIG. 5.

Glycolate concentrations for each 24-h sampling period. The data for three consecutive days in week 5 (5-1, 5-2, and 5-3) are indicated separately by the dashed lines and open symbols. Sample times were normalized to the sunrise time for the day. The error bars indicate the standard deviations for triplicate measurements of one sample per time point.