Analysis of subfossil molecular remains of purple sulfur bacteria in a lake sediment

Abstract

Molecular remains of purple sulfur bacteria (Chromatiaceae) were detected in Holocene sediment layers of a meromictic salt lake (Mahoney Lake, British Columbia, Canada). The carotenoid okenone and bacteriophaeophytin a were present in sediments up to 11,000 years old. Okenone is specific for only a few species of Chromatiaceae, including Amoebobacter purpureus, which presently predominates in the chemocline bacterial community of the lake. With a primer set specific for Chromatiaceae in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of four different Chromatiaceae species were retrieved from different depths of the sediment. One of the sequences, which originated from a 9, 100-year-old sample, was 99.2% identical to the 16S rRNA gene sequence of A. purpureus ML1 isolated from the chemocline. Employing primers specific for A. purpureus ML1 and dot blot hybridization of the PCR products, the detection limit for A. purpureus ML1 DNA could be lowered to 0.004% of the total community DNA. With this approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that A. purpureus ML1 constituted at least a part of the ancient purple sulfur bacterial community. The concentrations of A. purpureus DNA and okenone in the sediment were not correlated, and the ratio of DNA to okenone was much lower in the subfossil sediment layers (2.7 . 10(-6)) than in intact cells (1.4). This indicates that degradation rates are significantly higher for genomic DNA than for hydrocarbon cell constituents, even under anoxic conditions and at the very high sulfide concentrations present in Mahoney Lake.

FIG. 1

16S rRNA gene fragments from three strains of Chromatiaceae from the extant bacterial community in the chemocline and from samples from 10 different sediment layers of Mahoney Lake were amplified with primers Chr986f and GC1392r. An additional reaction mixture containing 1 μl of the DNA extraction control (see text) was included in the PCR. In two of the reactions, primer dimers formed during PCR (i.e., the 100-bp-long fragments in 5,580- and 8,220-year-old samples).

FIG. 2

Separation of the 16S rRNA gene fragments depicted in Fig. 1 on a DGGE gel. A negative image of an ethidium bromide-stained gel is shown. Percentages on the left denote concentrations of denaturant in the gel. Arrows point to the melting position of 16S rRNA gene fragment of A. purpureus ML1.

FIG. 3

Phylogenetic tree containing all available sequences of Chromatiaceae and the sequences obtained from the pelagial zone and sediment of Mahoney Lake. Shaded boxes indicate strains which contain the carotenoid okenone. y., years.

FIG. 4

Analysis of the melting behavior of 16S rRNA gene fragments generated by PCR with primers Ap454f and 907r. To obtain a visible amount of the DNA fragment and to introduce the GC clamp, the PCR products were reamplified with primers ApGC454f and 907r. A negative image of an ethidium bromide-stained DGGE gel is shown.

FIG. 5

Quantification of A. purpureus ML1 DNA in the Holocene sediment layers by amplification with primers Ap454f and 907r and dot blot hybridization. A digitized image of an exposed Lumi-Film is shown. Twenty-five nanograms of genomic DNA was added to the PCRs for T. winogradskyi DSMZ 6702^T (Trv.), T. roseopersicina ML2 (Tca.), and E. coli. For amplification of the chemocline sample, only 10 pg was used. Control denotes the amplification product of 1 μl of the extraction control (see text). For the detection of samples from subfossil sediments, 25 ng of extracted DNA was used. Sample numbering corresponds to that in Table 1. The different amounts of genomic DNA from A. purpureus ML1 used for calibration of the dot blot are given in picograms.

FIG. 6

Vertical distribution of BPh a (Bph a) and okenone expressed in milligrams per gram (dry weight) [mg (g d.w.)⁻¹] (A) and total DNA and DNA of A. purpureus ML1 (Amb. purpureus) in the sediment of Mahoney Lake. For direct comparison, the concentrations of okenone (dotted line) are also depicted in panel B. Total DNA is expressed in micrograms per gram (dry weight) [μg (g d.w.)⁻¹], and A. purpureus DNA is expressed in nanograms per gram (dry weight) [ng (g d.w.)⁻¹].

FIG. 7

Correlation between amounts of total DNA and DNA of A. purpureus ML1. Dotted lines delineate 95% confidence interval of second-order nonlinear regression. d.w., dry weight.

FIG. 8

Relative proportion of A. purpureus ML1 DNA of the total subfossil DNA compared to the range of fragment lengths of the subfossil DNA.