Novel, attached, sulfur-oxidizing bacteria at shallow hydrothermal vents possess vacuoles not involved in respiratory nitrate accumulation

Abstract

Novel, vacuolate sulfur bacteria occur at shallow hydrothermal vents near White Point, Calif. There, these filaments are attached densely to diverse biotic and abiotic substrates and extend one to several centimeters into the surrounding environment, where they are alternately exposed to sulfidic and oxygenated seawater. Characterizations of native filaments collected from this location indicate that these filaments possess novel morphological and physiological properties compared to all other vacuolate bacteria characterized to date. Attached filaments, ranging in diameter from 4 to 100 microm or more, were composed of cylindrical cells, each containing a thin annulus of sulfur globule-filled cytoplasm surrounding a large central vacuole. A near-complete 16S rRNA gene sequence was obtained and confirmed by fluorescent in situ hybridization to be associated only with filaments having a diameter of 10 microm or more. Phylogenetic analysis indicates that these wider, attached filaments form within the gamma proteobacteria a monophyletic group that includes all previously described vacuolate sulfur bacteria (the genera Beggiatoa, Thioploca, and Thiomargarita) and no nonvacuolate genera. However, unlike for all previously described vacuolate bacteria, repeated measurements of cell lysates from samples collected over 2 years indicate that the attached White Point filaments do not store internal nitrate. It is possible that these vacuoles are involved in transient storage of oxygen or contribute to the relative buoyancy of these filaments.

FIG 1

(A) White Point filament, stained with FITC, showing large internal vacuoles. Bar, 20 μm. (B) White Point filaments hybridized with White Point filament-specific probe WPF464 in 20% formamide. Bar, 50 μm. (C) Light micrograph of large White Point filament. Bar, 50 μm. (D) Light micrograph of White Point filaments forming a rosette. Bar, 40 μm.

FIG 2

Relationship of frequency to diameter for VAF collected from rocks at White Point vents (water depth, ca. 6 m). Filaments less than 10 μm in diameter (white bars) did not hybridize with any fluorescent probe, including WPF464, while all wider, attached filaments (black bars) positively hybridized with WPF464. Suspensions were collected in July 2001 (A) and February 2002 (B).

FIG 3

Relationship of fluorescence intensity (pixel strength maximum, 255) of bound probes to hybridization stringency. (A) White Point attached filaments hybridized with gamma and beta proteobacterial probes. Results with the GAM42a probe with filaments with diameters of <10 μm (▪), 10 to 29 μm (▴), and 30 to 70 μm (▵) are shown. The average signal for filaments of all widths with the BET42a probe (○) is also shown. Similarly, low fluorescence signals were recorded at 488 and 586 nm for no-probe controls (background fluorescence) with filaments of all widths. (B) Carmel Canyon Beggiatoa sp. hybridized with gamma and beta proteobacterial probes, including the GAM42a probe (•), the BET42a probe (▪), the no-probe control excited at 586 nm (○), and the no-probe control excited at 488 nm (□). (C) White Point attached filaments and Carmel Canyon Beggiatoa sp. hybridized with the VSO673 probe specific for large, nitrate-accumulating, vacuolate, sulfur-oxidizing bacteria. Results for White Point attached filaments with diameters of 10 to 29 μm (▴) and 30 to 70 μm (▵) are shown, as well as Carmel Canyon Beggiatoa sp. (•), the Carmel Canyon Beggiatoa sp. no-probe control (○), and the White Point attached filament no-probe control (10 to 70 μm in diameter) (□). (D) Results for White Point attached filaments and Carmel Canyon Beggiatoa sp. hybridized with the WPF464 probe specific for White Point attached filaments are shown. Symbols are the same as for panel C.

FIG 4

Minimum evolutionary tree of the White Point VAF and diverse sulfur-oxidizing gamma proteobacteria, based on 16S rRNA gene sequences. Sequences of epsilon proteobacteria are included as an outgroup. The tree was constructed with an alignment of positions 126 to 1376 (E. coli numbering). Numbers on the nodes represent percentage bootstrap values greater than 50% (1,000 replicates). Accession numbers are shown.