Diversity and function of Chloroflexus-like bacteria in a hypersaline microbial mat: phylogenetic characterization and impact on aerobic respiration

Abstract

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and gamma-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.

FIG. 1.

Steady-state oxygen and sulfide profiles in Lake Chiprana microbial mats at different light intensities. (A) Oxygen concentration profiles with 0, 33, 83, 166, and 300 μmol photons m⁻² s⁻¹. The oxygen concentration reaches up to six times the air saturation level at higher light intensities, indicating that CLB in the photic zone have to cope with high and fluctuating oxygen concentrations. (B) Oxygen and sulfide (H₂S) concentration profiles at light intensities of 83 and 300 μmol photons m⁻² s⁻¹. Oxygen and sulfide cooccur in the zone between 1 and 1.5 mm deep, a zone where CLB are able to oxidize sulfide both phototrophically and chemotrophically, in the latter case using oxygen as the electron acceptor. μE, microeinsteins.

FIG. 2.

HPLC chromatograms at 440, 660, and 760 nm for a sample from the top 5 mm of the Lake Chiprana microbial mat. Identified pigment molecules, indicated by numbers, are described in Table 1. The main pigments are Chla (peak 17) and BChlc allomers (peaks 5, 6, 8, 12 to 14, and 16).

FIG. 3.

16S rRNA gene maximum parsimony tree for representative sequences obtained in this study (bold type) and sequences retrieved from the database. Sequences from this study in the tree represent groups of sequences that shared more than 96% identity; the numbers in parentheses indicate the numbers of sequences in the groups. Asterisks indicate known phototrophic species, and number signs indicate previously isolated and described species. The bootstrap values at the nodes are percentages based on 1,000 replications. Sequences from this study, as well as LO4675, AJ09636, and AJ309642, were excluded from the bootstrap analysis. Group I represents sequences recovered in this study, which formed a separate clade that included H. oregonesis (a chlorosome-less, BChla- but not BChlc- or BChld-producing species) and “Candidatus Chlorothrix” (chlorosome-containing, BChla- and BChlc-producing species); group II represents sequences obtained in this study that formed a clade with Chloroflexus, Chloronema, and Oscillochloris (chlorosome-containing, BChla- plus BChlc- or BChld-producing species). Groups III to VIII represent sequences from this study which cluster with sequences distantly related to the family Chloroflexaceae but are still in the phylum Chloroflexi.

FIG. 4.

Oxygen consumption in aerated axenic cultures of C. aurantiacus under different illumination conditions. (A) Alternating illumination with NIR light (25-W incandescent light bulb plus two 40-mA NIR LEDs [710 to 770 nm]) and darkness. (B) Alternating illumination with VIS light (two VIS LEDs [400 to 700 nm]) and darkness, as well as the formaldehyde-killed control. See Table 3 for calculated specific respiration rates.

FIG. 5.

Steady-state oxygen concentration profiles in the Lake Chiprana microbial mat under four different light conditions: (i) darkness, (ii) NIR light (two 40-mA NIR LEDs [710 to 770 nm]), (iii) VIS light (two VIS LEDs [400 to 700 nm]; 60 μmol photons m⁻² s⁻¹), and (iv) two NIR LEDs plus two VIS LEDs.