A cascade of sulfur transferases delivers sulfur to the sulfur-oxidizing heterodisulfide reductase-like complex.

A heterodisulfide reductase-like complex (sHdr) and novel lipoate-binding proteins (LbpAs) are central players of a wide-spread pathway of dissimilatory sulfur oxidation. Bioinformatic analysis demonstrate that the cytoplasmic sHdr-LbpA systems are always accompanied by sets of sulfur transferases (DsrE proteins, TusA, and rhodaneses). The exact composition of these sets may vary depending on the organism and sHdr system type. To enable generalizations, we studied model sulfur oxidizers from distant bacterial phyla, that is, Aquificota and Pseudomonadota. DsrE3C of the chemoorganotrophic Alphaproteobacterium Hyphomicrobium denitrificans and DsrE3B from the Gammaproteobacteria Thioalkalivibrio sp. K90mix, an obligate chemolithotroph, and Thiorhodospira sibirica, an obligate photolithotroph, are homotrimers that donate sulfur to TusA. Additionally, the hyphomicrobial rhodanese-like protein Rhd442 exchanges sulfur with both TusA and DsrE3C. The latter is essential for sulfur oxidation in Hm. denitrificans. TusA from Aquifex aeolicus (AqTusA) interacts physiologically with AqDsrE, AqLbpA, and AqsHdr proteins. This is particularly significant as it establishes a direct link between sulfur transferases and the sHdr-LbpA complex that oxidizes sulfane sulfur to sulfite. In vivo, it is unlikely that there is a strict unidirectional transfer between the sulfur-binding enzymes studied. Rather, the sulfur transferases form a network, each with a pool of bound sulfur. Sulfur flux can then be shifted in one direction or the other depending on metabolic requirements. A single pair of sulfur-binding proteins with a preferred transfer direction, such as a DsrE3-type protein towards TusA, may be sufficient to push sulfur into the sink where it is further metabolized or needed.

Fe/S proteins in microbial sulfur oxidation.

Iron-sulfur clusters serve as indispensable cofactors within proteins across all three domains of life. Fe/S clusters emerged early during the evolution of life on our planet and the biogeochemical cycle of sulfur is one of the most ancient and important element cycles. It is therefore no surprise that Fe/S proteins have crucial roles in the multiple steps of microbial sulfur metabolism. During dissimilatory sulfur oxidation in prokaryotes, Fe/S proteins not only serve as electron carriers in several steps, but also perform catalytic roles, including unprecedented reactions. Two cytoplasmic enzyme systems that oxidize sulfane sulfur to sulfite are of particular interest in this context: The rDsr pathway employs the reverse acting dissimilatory sulfite reductase rDsrAB as its key enzyme, while the sHdr pathway utilizes polypeptides resembling the HdrA, HdrB and HdrC subunits of heterodisulfide reductase from methanogenic archaea. Both pathways involve components predicted to bind unusual noncubane Fe/S clusters acting as catalysts for the formation of disulfide or sulfite. Mapping of Fe/S cluster machineries on the sulfur-oxidizing prokaryote tree reveals that ISC, SUF, MIS and SMS are all sufficient to meet the Fe/S cluster maturation requirements for operation of the sHdr or rDsr pathways. The sHdr pathway is dependent on lipoate-binding proteins that are assembled by a novel pathway, involving two Radical SAM proteins, namely LipS1 and LipS2. These proteins coordinate sulfur-donating auxiliary Fe/S clusters in atypical patterns by three cysteines and one histidine and act as lipoyl synthases by jointly inserting two sulfur atoms to an octanoyl residue. This article is part of a Special Issue entitled: Biogenesis and Function of Fe/S proteins.

In the Alphaproteobacterium Hyphomicrobium denitrificans SoxR Serves a Sulfane Sulfur-Responsive Repressor of Sulfur Oxidation.

In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of genes for enzymes involved in sulfur oxidation is needed to adjust metabolic flux to environmental conditions. However, little is known about the sensing and response to inorganic sulfur compounds such as thiosulfate in sulfur-oxidizing bacteria. In the Alphaproteobacterium Hyphomicrobium denitrificans, one strategy is the use of the ArsR-SmtB-type transcriptional regulator SoxR. We show that this homodimeric repressor senses sulfane sulfur and that it is crucial for the expression not only of sox genes encoding the components of a truncated periplasmic thiosulfate-oxidizing enzyme system but also of several other sets of genes for enzymes of sulfur oxidation. DNA binding and transcriptional regulatory activity of SoxR are controlled by polysulfide-dependent cysteine modification. The repressor uses the formation of a sulfur bridge between two conserved cysteines as a trigger to bind and release DNA and can also form a vicinal disulfide bond to orchestrate a response to oxidizing conditions. The importance of the sulfur bridge forming cysteines was confirmed by site-directed mutagenesis, mass spectrometry, and gel shift assays. In vivo, SoxR interacts directly or indirectly with a second closely related repressor, sHdrR.

HMSS2: An advanced tool for the analysis of sulphur metabolism, including organosulphur compound transformation, in genome and metagenome assemblies.

The global sulphur cycle has implications for human health, climate change, biogeochemistry and bioremediation. The organosulphur compounds that participate in this cycle not only represent a vast reservoir of sulphur but are also used by prokaryotes as sources of energy and/or carbon. Closely linked to the inorganic sulphur cycle, it involves the interaction of prokaryotes, eukaryotes and chemical processes. However, ecological and evolutionary studies of the conversion of organic sulphur compounds are hampered by the poor conservation of the relevant pathways and their variation even within strains of the same species. In addition, several proteins involved in the conversion of sulphonated compounds are related to proteins involved in sulphur dissimilation or turnover of other compounds. Therefore, the enzymes involved in the metabolism of organic sulphur compounds are usually not correctly annotated in public databases. To address this challenge, we have developed HMSS2, a profiled Hidden Markov Model-based tool for rapid annotation and synteny analysis of organic and inorganic sulphur cycle proteins in prokaryotic genomes. Compared to its previous version (HMS-S-S), HMSS2 includes several new features. HMM-based annotation is now supported by nonhomology criteria and covers the metabolic pathways of important organosulphur compounds, including dimethylsulphoniopropionate, taurine, isethionate, and sulphoquinovose. In addition, the calculation speed has been increased by a factor of four and the available output formats have been extended to include iTol compatible data sets, and customized sequence FASTA files.

Identification of a novel lipoic acid biosynthesis pathway reveals the complex evolution of lipoate assembly in prokaryotes.

Lipoic acid is an essential biomolecule found in all domains of life and is involved in central carbon metabolism and dissimilatory sulfur oxidation. The machineries for lipoate assembly in mitochondria and chloroplasts of higher eukaryotes, as well as in the apicoplasts of some protozoa, are all of prokaryotic origin. Here, we provide experimental evidence for a novel lipoate assembly pathway in bacteria based on a sLpl(AB) lipoate:protein ligase, which attaches octanoate or lipoate to apo-proteins, and 2 radical SAM proteins, LipS1 and LipS2, which work together as lipoyl synthase and insert 2 sulfur atoms. Extensive homology searches combined with genomic context analyses allowed us to precisely distinguish between the new and established pathways and map them on the tree of life. This not only revealed a much wider distribution of lipoate biogenesis systems than expected, in particular, the novel sLpl(AB)-LipS1/S2 pathway, and indicated a highly modular nature of the enzymes involved, with unforeseen combinations, but also provided a new framework for the evolution of lipoate assembly. Our results show that dedicated machineries for both de novo lipoate biogenesis and scavenging from the environment were implemented early in evolution and that their distribution in the 2 prokaryotic domains was shaped by a complex network of horizontal gene transfers, acquisition of additional genes, fusions, and losses. Our large-scale phylogenetic analyses identify the bipartite archaeal LplAB ligase as the ancestor of the bacterial sLpl(AB) proteins, which were obtained by horizontal gene transfer. LipS1/S2 have a more complex evolutionary history with multiple of such events but probably also originated in the domain archaea.

HMS-S-S: A tool for the identification of Sulphur metabolism-related genes and analysis of operon structures in genome and metagenome assemblies.

Sulphur compounds are used in a variety of biological processes including respiration and photosynthesis. Sulphide and sulphur compounds of intermediary oxidation state can serve as electron donors for lithotrophic growth while sulphate, thiosulphate and sulphur are used as electron acceptors in anaerobic respiration. The biochemistry underlying the manifold transformations of inorganic sulphur compounds occurring in sulphur metabolizing prokaryotes is astonishingly complex and knowledge about it has immensely increased over the last years. The advent of next-generation sequencing approaches as well as the significant increase of data availability in public databases has driven focus of environmental microbiology to probing the metabolic capacity of microbial communities by analysis of this sequence data. To facilitate these analyses, we created HMS-S-S, a comprehensive equivalogous hidden Markov model (HMM)-supported tool. Protein sequences related to sulphur compound oxidation, reduction, transport and intracellular transfer are efficiently detected and related enzymes involved in dissimilatory sulphur oxidation as opposed to sulphur compound reduction can be confidently distinguished. HMM search results are coupled to corresponding genes, which allows analysis of co-occurrence, synteny and genomic neighbourhood. The HMMs were validated on an annotated test data set and by cross-validation. We also proved its performance by exploring meta-assembled genomes isolated from samples from environments with active sulphur cycling, including members of the cable bacteria, novel Acidobacteria and assemblies from a sulphur-rich glacier, and were able to replicate and extend previous reports.

The functional diversity of the prokaryotic sulfur carrier protein TusA.

Persulfide groups participate in a wide array of biochemical pathways and are chemically very versatile. The TusA protein has been identified as a central element supplying and transferring sulfur as persulfide to a number of important biosynthetic pathways, like molybdenum cofactor biosynthesis or thiomodifications in nucleosides of tRNAs. In recent years, it has furthermore become obvious that this protein is indispensable for the oxidation of sulfur compounds in the cytoplasm. Phylogenetic analyses revealed that different TusA protein variants exists in certain organisms, that have evolved to pursue specific roles in cellular pathways. The specific TusA-like proteins thereby cannot replace each other in their specific roles and are rather specific to one sulfur transfer pathway or shared between two pathways. While certain bacteria like Escherichia coli contain several copies of TusA-like proteins, in other bacteria like Allochromatium vinosum a single copy of TusA is present with an essential role for this organism. Here, we give an overview on the multiple roles of the various TusA-like proteins in sulfur transfer pathways in different organisms to shed light on the remaining mysteries of this versatile protein.