Comparative genomic analysis of fruiting body formation in Myxococcales.

Genetic programs underlying multicellular morphogenesis and cellular differentiation are most often associated with eukaryotic organisms, but examples also exist in bacteria such as the formation of multicellular, spore-filled fruiting bodies in the order Myxococcales. Most members of the Myxococcales undergo a multicellular developmental program culminating in the formation of spore-filled fruiting bodies in response to starvation. To gain insight into the evolutionary history of fruiting body formation in Myxococcales, we performed a comparative analysis of the genomes and transcriptomes of five Myxococcales species, four of these undergo fruiting body formation (Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, and Haliangium ochraceum) and one does not (Anaeromyxobacter dehalogenans). Our analyses show that a set of 95 known M. xanthus development-specific genes--although suffering from a sampling bias--are overrepresented and occur more frequently than an average M. xanthus gene in S. aurantiaca, whereas they occur at the same frequency as an average M. xanthus gene in S. cellulosum and in H. ochraceum and are underrepresented in A. dehalogenans. Moreover, genes for entire signal transduction pathways important for fruiting body formation in M. xanthus are conserved in S. aurantiaca, whereas only a minority of these genes are conserved in A. dehalogenans, S. cellulosum, and H. ochraceum. Likewise, global gene expression profiling of developmentally regulated genes showed that genes that upregulated during development in M. xanthus are overrepresented in S. aurantiaca and slightly underrepresented in A. dehalogenans, S. cellulosum, and H. ochraceum. These comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are highly similar and significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. Thus, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxococcales and strongly suggest that the genetic program underlying fruiting body formation in different Myxococcales is not conserved. The evolutionary implications of this finding are discussed.

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe-4S] cluster.

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CX(n)CCGX(m)CXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe-2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), (57)Fe electron-nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe-4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe-4S](2+) cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with g (zyx) = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with g (zyx) = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S(3)(O/N)(1) coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron-sulfur cluster and the zinc site.

Bioinformatics and experimental analysis of proteins of two-component systems in Myxococcus xanthus.

Proteins of two-component systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria and in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus, TCS are important for normal motility and fruiting body formation and sporulation. Here we analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. Two hundred seventy-two TCS genes were identified, 251 of which are not part of che gene clusters. We report that the TCS genes are unusually organized, with 55% being orphan and 16% in complex gene clusters whereas only 29% display the standard paired gene organization. Hybrid histidine protein kinases and histidine protein kinases predicted to be localized to the cytoplasm are overrepresented among proteins encoded by orphan genes or in complex gene clusters. Similarly, response regulators without output domains are overrepresented among proteins encoded by orphan genes or in complex gene clusters. The most frequently occurring output domains in response regulators are involved in DNA binding and cyclic-di-GMP metabolism. Our analyses suggest that TCS encoded by orphan genes and complex gene clusters are functionally distinct from TCS encoded by paired genes and that the connectivity of the pathways made up of TCS encoded by orphan genes and complex gene clusters is different from that of pathways involving TCS encoded by paired genes. Experimentally, we observed that orphan TCS genes are overrepresented among genes that display altered transcription during fruiting body formation. The systematic analysis of the 25 orphan genes encoding histidine protein kinases that are transcriptionally up-regulated during development showed that 2 such genes are likely essential for viability and identified 7 histidine protein kinases, including 4 not previously characterized that have important function in fruiting body formation or spore germination.

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.

Heterodisulfide reductase from methanogenic archaea: a new catalytic role for an iron-sulfur cluster.

Heterodisulfide reductase (HDR) from methanogenic archaea is an iron-sulfur protein that catalyzes reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Via the characterization of a paramagnetic reaction intermediate generated upon oxidation of the enzyme in the presence of coenzyme M, the enzyme was shown to contain a [4Fe-4S] cluster in its active site that catalyzes reduction of the disulfide substrate in two one-electron reduction steps. The formal thiyl radical generated by the initial one-electron reduction of the disulfide is stabilized via reduction and coordination of the resultant thiol to the [4Fe-4S] cluster.