Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.     

Photosynthesis Genes and LH1 Proteins of Roseospirillum Parvum 930I,a Purple Non-Sulfur Bacterium with Unusual Spectral Properties

The purple bacterium Roseospirillum(Rss.)parvum 930I, like most other purple bacteria, contains bacteriochlorophyll (BChl)-a as a LH chromophore, but exhibits an extremely red-shifted Q _y absorption maximum centered at 909 nm. The puf operon encoding the LH1 and other pigment-binding proteins was cloned and sequenced, revealing the gene structure pufBALMC. Comparative analysis of predicted amino acid sequences of the - and -core LH polypeptides (PufA and PufB) revealed five amino-acid substitutions at positions that are highly conserved in other purple bacteria. In the primary structure, these residues are in close proximity to the BChl-a ligating histidine residue ( ⁺³, ⁺⁵, ⁺⁶ and ^–4, ⁺⁹, with respect to the central histidine, His⁰). The nature of the enormous red-shifts to the near-infrared region of the light were attributed to those residues, with special emphasis to cysteine residues rarely present in other purple bacterial antenna polypeptides. Three-dimensional structural models of the Rss. parvum LH1- and - polypeptides suggest that the thiol groups of Cys⁺³ and Cys^–4 are oriented toward the BChl-a macrocycle in the native antenna complex. Because of their predicted close proximity to BChl, these thiol groups are likely to modulate the spectral properties of the LH1 complex. Phylogenetic comparisons of LH1 polypeptides indicate that Rss. parvum-like amino-acid substitutions in proteobacterial LH1 complexes arose independently during evolution.This revised version was published online in October 2005 with corrections to the Cover Date.     

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 °C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriopheophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in “Chlorochromatium aggregatum” and “Pelochromatium roseum” could be demonstrated for the first time. Received: 26 October 1998 / Accepted: 7 January 1999     

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and β-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, β-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species. Received: 22 January 1999 / Accepted: 28 April 1999     

The ecological niche of the consortium “Pelochromatium roseum”

A dense accumulation of the phototrophic consortium “Pelochromatium roseum” in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per “P. roseum” a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 μmol quanta m^–2 s^–1) and low sulfide concentrations (0–100 μM). In dialysis cultures of “P. roseum”, bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 μmol quanta m^–2 s^–1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m^–3, which was much higher than that of ambient chemocline water (995.8 kg m^–3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves. Received: 27 May 1997 / Accepted: 16 September 1997     

Phylogeny and molecular fingerprinting of green sulfur bacteria

The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla. The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030. Similarity values > 90.1% and K_nuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria. As a group, these bacteria represent an isolated branch within the eubacterial radiation. In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness. While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species. Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids. Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated. The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis. In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR. The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria. Received: 26 August 1996 / Accepted: 8 January 1997