Most of the processes in the inorganic sulfur metabolism of prokaryotes, especially those involving elemental sulfur, are poorly described despite their significance for cellular growth and their impact on the environment. This incomplete knowledge is in part due to the limitations posed by traditional genetic and enzymological approaches.

We investigate the inorganic sulfur metabolism in the green sulfur bacterium Chlorobium tepidum based on a genomics approach as a model for the inorganic sulfur metabolism in both phototrophic and chemotrophic prokaryotes. Based on our genome analyses and the limited biochemical and physiological information available on the inorganic sulfur metabolism in green sulfur bacteria, it is obvious that novel enzymes and pathways remain to be discovered. Our research approaches this problem by combining bioinformatic analyses with targeted gene inactivations, transcriptome analyses, and biochemical and physiological studies.

Figure 1. (A) Cross-section of a stratified fresh-water lake. (B) Simplified scheme of the microbial transformations of inorganic sulfur compounds.

Figure 2. Simplified scheme of sulfide, thiosulfate, and elemental sulfur oxidation in Chl. tepidum.

Empirical characterizations of substrate preferences and conversions in green sulfur bacteria have been performed for decades. However, only a few enzymes have been isolated and characterized from these bacteria and genetic analyses are even scarcer. One important and convenient advantage in using Chl. tepidum as the model organism for sulfur oxidation in green sulfur bacteria is that this organism can perform most, if not all, of the sulfur metabolism represented by green sulfur bacteria. Based on analyses of the genome sequence of Chl. tepidum a model for the inorganic sulfur metabolism in this organism has been proposed (Fig. 3).

Figure 3. Proposed enzymes and pathways of inorganic sulfur metabolism in Chl. tepidum (modified from Eisen et al. 2002 PNAS 99:9509).

The limited metabolic capabilities of green sulfur bacteria are reflected by their small genomes that generally are between 2 and 3 Mbp. Analyses of the Chl. tepidum genome confirm that this organism has limited metabolic capabilities and limited transcriptional regulation. Genes involved in photosynthesis in this organism are scattered throughout the genome. Importantly however, genes thought to be involved in inorganic sulfur metabolism are numerous and clustered, which suggests enzymatic versatility and transcriptional regulation.

Gene ortholog neighborhoods
One approach to identify genes that are involved in inorganic sulfur metabolism is to investigate the gene contents in many sulfur-metabolizing organisms simultaneously. One such analysis shows that homologs of the putative polysulfide reductase CT0496 in Chl. tepidum are present in several other green sulfur bacteria and sulfur-metabolizing archaea and bacteria (Fig. 4).

Figure 4. Gene neighborhoods of six CT0496 top hits (putative polysulfide reductase; marked in red) among 414 genome-sequenced organisms. (Calculated using the Integrated Microbial Genomes server from the Joint Genome Institute.)

Whole-genome phylogenetic profile analysis
Calculating phylogenetic profiles can help predict protein functions by correlating the inheritance of proteins across different species (Pellegrini et al. 1999 PNAS 96:4285). This is because proteins that have similar profiles strongly tend to be functionally linked. One such analysis of the genome of the green sulfur bacterium Chlorobium chlorochromatii has identified a cluster of 9 heterodisulfide reductase-related proteins and iron-sulfur proteins (Fig. 5). All these 9 proteins are present in 3 prokaryotes (marked in yellow) that metabolize elemental sulfur. Thus, these 9 proteins may be involved in elemental sulfur metabolism.

Figure 5. Section of a whole-genome phylogenetic profile analysis of the green sulfur bacterium Chlorobium chlorochromatii against 182 completely sequenced microbial genomes.

Phylogenetic analyses
Some of the more exciting aspects of microbial evolution are how genes are transferred between organisms (lateral gene transfer) and how genes duplicate and evolve into new functions within the same organism. Phylogenetic analyses involving fully sequenced genomes sometimes reveal such phenomena. SoxA and SoxY are both components of an enzyme system that oxidizes inorganic sulfur compounds in thiotrophic prokaryotes like green sulfur bacteria (Fig. 3). Whereas only one type of SoxA is present in green sulfur bacteria, two types of SoxY are present (Fig. 6).

Figure 6. Phylogenetic trees of two proteins (SoxA and SoxY) involved in inorganic sulfur oxidation in thiotrophic prokaryotes.

REVIEW Frigaard, N.-U. and Bryant, D. A. (2004) Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria. Archives of Microbiology 182: 265-276. [PubMed]

REVIEW Frigaard, N.-U., Gomez Maqueo Chew, A., Li, H., Maresca, J. A. and Bryant, D. A. (2003) Chlorobium tepidum: Insights into the structure, physiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence. Photosynthesis Research 78: 93-117. [Kluwer]