Genome Sequence and Characterization of a Xanthorhodopsin-Containing, Aerobic Anoxygenic Phototrophic Rhodobacter Species, Isolated from Mesophilic Conditions at Yellowstone National Park.

The genus Rhodobacter consists of purple nonsulfur photosynthetic alphaproteobacteria known for their diverse metabolic capabilities. Here, we report the genome sequence and initial characterization of a novel Rhodobacter species, strain M37P, isolated from Mushroom hot spring runoff in Yellowstone National Park at 37 °C. Genome-based analyses and initial growth characteristics helped to define the differentiating characteristics of this species and identified it as an aerobic anoxygenic phototroph (AAP). This is the first AAP identified in the genus Rhodobacter. Strain M37P has a pinkish-red pigmentation that is present under aerobic dark conditions but disappears under light incubation. Whole genome-based analysis and average nucleotide identity (ANI) comparison indicate that strain M37P belongs to a specific clade of recently identified species that are genetically and physiologically unique from other representative Rhodobacter species. The genome encodes a unique xanthorhodopsin, not found in any other Rhodobacter species, which may be responsible for the pinkish-red pigmentation. These analyses indicates that strain M37P is a unique species that is well-adapted to optimized growth in the Yellowstone hot spring runoff, for which we propose the name Rhodobacter calidifons sp. nov.

Comparative Genome Analysis of the Photosynthetic Betaproteobacteria of the Genus Rhodocyclus: Heterogeneity within Strains Assigned to Rhodocyclus tenuis and Description of Rhodocyclus gracilis sp. nov. as a New Species.

The genome sequences for Rhodocyclus purpureus DSM 168T and four strains assigned to Rhodocyclus tenuis (DSM 110, DSM 111, DSM 112, and IM 230) have been determined. One of the strains studied (IM 230) has an average nucleotide identity (ANI) of 97% to the recently reported genome of the type strain DSM 109 of Rcy. tenuis and is regarded as virtually identical at the species level. The ANI of 80% for three other strains (DSM 110, DSM 111, DSM 112) to the type strain of Rcy. tenuis points to a differentiation of these at the species level. Rcy. purpureus is equidistant from Rcy. tenuis and the new species, based on both ANI (78-80%) and complete proteome comparisons (70% AAI). Strains DSM 110, DSM 111, and DSM 112 are very closely related to each other based on ANI, whole genome, and proteome comparisons but clearly distinct from the Rcy. tenuis type strain DSM 109. In addition to the whole genome differentiation, these three strains also contain unique genetic differences in cytochrome genes and contain genes for an anaerobic cobalamin synthesis pathway that is lacking from both Rcy. tenuis and Rcy. purpureus. Based on genomic and genetic differences, these three strains should be considered to represent a new species, which is distinctly different from both Rcy. purpureus and Rcy. tenuis, for which the new name Rhodocyclus gracilis sp. nov. is proposed.

Genomic Comparison, Phylogeny and Taxonomic Reevaluation of the Ectothiorhodospiraceae and Description of Halorhodospiraceae fam. nov. and Halochlorospira gen. nov.

The Ectothiorhodospiraceae family represents purple sulfur bacteria of the Gammaproteobacteria found primarily in alkaline soda lakes of moderate to extremely high salinity. The main microscopically visible characteristic separating them from the Chromatiaceae is the excretion of the intermediate elemental sulfur formed during oxidation of sulfide prior to complete oxidation to sulfate rather than storing it in the periplasm. We present a comparative study of 38 genomes of all species of phototrophic Ectothiorhodospiraceae. We also include a comparison with those chemotrophic bacteria that have been assigned to the family previously and critically reevaluate this assignment. The data demonstrate the separation of Halorhodospira species in a major phylogenetic branch distant from other Ectothiorhodospiraceae and support their separation into a new family, for which the name Halorhodospiraceae fam. nov. is proposed. In addition, the green-colored, bacteriochlorophyll-containing species Halorhodospira halochloris and Halorhodospira abdelmalekii were transferred to the new genus Halochlorospira gen. nov. of this family. The data also enable classification of several so far unclassified isolates and support the separation of Ectothiorhodospira shaposhnikovii and Ect. vacuolata as well as Ect. mobilis and Ect. marismortui as distinct species.

Phylogenetic relationship of phototrophic heliobacteria and systematic reconsideration of species and genus assignments based on genome sequences of eight species.

The draft genome sequences of five species of named phototrophic heliobacteria in the order Clostridiales were determined. Whole genome phylogenetic and average nucleotide identity comparison for the heliobacteria suggests that Heliobacterium chlorum and Heliobacillus mobilis are closely related to one another and belong to the same genus. The three species Heliobacterium modesticaldum, Heliobacterium undosum and Heliobacterium gestii all belong in the same genus, but are more divergent from Hbt. chlorum and belong in a separate genus, which we suggest to be called Heliomicrobium. Heliorestis convoluta is properly recognized to be in the same genus as Heliorestis acidaminivorans. Heliophilum fasciatum is clearly unlike any other and rightfully belongs in a separate genus.

The genome sequence of the giant phototrophic gammaproteobacterium Thiospirillum jenense gives insight into its physiological properties and phylogenetic relationships.

In a conserved culture of the purple sulfur bacterium Thiospirillum jenense DSM216T, cells of this species were easily recognized by cell morphology, large-size spirilla and visible flagellar tuft. The Tsp. jenense genome is 3.22 Mb in size and has a GC content of 48.7 mol%. It was readily identified as a member of the Chromatiaceae by the complement of proteins in its genome. A whole genome comparison clearly placed Tsp. jenense near Thiorhodovibrio and Rhabdochromatium species and somewhat more distant from Thiohalocapsa and Halochromatium species. This relationship was also found with the sequences of the photosynthetic reaction center protein PufM. The genome sequence supported important properties of this bacterium: the presence of ribulose-bisphosphate carboxylase and enzymes of the Calvin cycle of autotrophic carbon dioxide fixation but the absence of carboxysomes, an incomplete tricarboxylic acid cycle and the lack of malate dehydrogenase, the presence of a sulfur oxidation pathway including adenylylsulfate reductase (aprAB) but absence of assimilatory sulfate reduction, the presence of hydrogenase (hoxHMFYUFE), nitrogenase and a photosynthetic gene cluster (pufBALMC). The FixNOP type of cytochrome oxidase was notably lacking, which may be the reason that renders the cells highly sensitive to oxygen. Two minor phototrophic contaminants were found using metagenomic binning: one was identified as a strain of Rhodopseudomonas palustris and the second one has an average nucleotide identity of 82% to the nearest neighbor Rhodoferax antarcticus. It should be considered as a new species of this genus and Rhodoferax jenense is proposed as the name.

Genomic and genetic sequence information of strains assigned to the genus Rhodopseudomonas reveal the great heterogeneity of the group and identify strain Rhodopseudomonas palustris DSM 123T as the authentic type strain of this species.

The genus Rhodopseudomonas, containing purple nonsulfur photosynthetic Proteobacteria, has a number of strains that belong to different species, although many of them are collectively called Rhodopseudomonas palustris. The type species R. palustris and closely related species are the focus of this paper. The comparison of available genome sequences indicate that the following Rhodopseudomonas species are well recognized: R. palustris (strains ATH 2.1.6T=DSM 123T=NBRC 100419T and BisB5), Rhodopseudomonas rutila (strains R1T, DSM 126, CGA009, ATH 2.1.37, Eli 1980, ATCC 17001 and TIE1), Rhodopseudomonas pentothenatexigens JA575T and Rhodopseudomonas faecalis JCM 11668T. Other strains for which genome sequences are available are distinct from these four species. Evidence is presented that R. palustris strain ATH 2.1.6T-KCM as obtained directly from the van Niel collection by one of us (T.E.M.) is identical to the DSMZ deposit DSM 123T of ATH 2.1.6T, but not to the deposit at ATCC 17001. The amino acid sequences of the cytochromes C2 and C556 from R. palustris strain ATH 2.1.6T-KCM are in complete agreement with the translated genome sequences of R. palustris DSM 123T. In addition, the 16S rRNA gene sequence of R. palustris NBRC 100419T completely matches that of strain DSM 123T. In conclusion, the type strain of R. palustris ATH 2.1.6T is correctly represented by DSM 123T and NBRC 100419T. However, the deposit at ATCC 17001 has properties that do not conform with properties of authentic R. palustris, but rather indicate that this is a strain of R. rutila. The previously suggested assignment of the type strain of R. palustris DSM 123T to the new species R. pseudopalustris was incorrect because strain DSM 123T is the authentic type strain of R. palustris.

GHP, a new c-type green heme protein from Halochromatium salexigens and other proteobacteria.

We have isolated a minor soluble green-colored heme protein (GHP) from the purple sulfur bacterium, Halochromatium salexigens, which contains a c-type heme. A similar protein has also been observed in the purple bacteria Allochromatium vinosum and Rhodopseudomonas cryptolactis. This protein has wavelength maxima at 355, 420, and 540 nm and remains unchanged upon addition of sodium dithionite or potassium ferricyanide, indicating either an unusually low or high redox potential, respectively. The amino-acid sequence indicates one heme per peptide chain of 72 residues and reveals weak similarity to the class I cytochromes. The usual sixth heme ligand methionine in these proteins appears to be replaced by a cysteine in GHP. Only one known cytochrome has a cysteine sixth ligand, SoxA (cytochrome c-551) from thiosulfate-oxidizing bacteria, which is low-spin and has a high redox potential because of an un-ionized ligand. The native size of GHP is 34 kDa, its subunit size is 11 kDa, and the net charge is -12, accounting for its very acidic nature. A database search of complete genome sequences reveals six homologs, all hypothetical proteins, from Oceanospirillum sp., Magnetococcus sp., Thiobacillus denitrificans, Dechloromonas aromatica, Thiomicrospira crunogena and Methylobium petroleophilum, with sequence identities of 35-64%. The genetic context is different for each species, although the gene for GHP is transcriptionally linked to several other genes in three out of the six species. These genes, coding for an RNAse, a protease/chaperone, a GTPase, and pterin-4a-carbinolamine dehydratase, appear to be functionally related to stress response and are linked in at least 10 species.

EPR analysis of multiple forms of [4Fe-4S](3+) clusters in HiPIPs.

The electron paramagnetic resonance (EPR) spectrum from the [4Fe-4S](3+) cluster in several high-potential iron-sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe-4S](3+) structure the (Fe-Fe)(5+) mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe-4S](3+) paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe-4S](3+) model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.

Sensitive circular dichroism marker for the chromophore environment of photoactive yellow protein: assignment of the 307 and 318 nm bands to the n --> pi* transition of the carbonyl.

The absorption and CD spectra of wild-type PYP, apo-PYP, and the mutants, E46Q and M100A, were measured between 250 and 550 nm. At neutral pH, the two very weak absorption bands of wild-type PYP at 307 and 318 nm (epsilon(max) = 600 +/- 100 M(-1) cm(-1) at 318 nm) are associated with quite strong positive CD bands (Deltaepsilon(max) approximately 6.8 M(-1) cm(-1)). Both sets of bands are absent in the apoprotein. On the basis of this evidence, we assign these optical signals to the n --> pi* transition of the oxygen of the carbonyl group of the 4-hydroxycinnamic acid chromophore, which is expected to be electric dipole forbidden but magnetic dipole allowed. The progression of narrow bands at 307 and 318 nm with a shoulder in the CD around 329 nm is due to vibrational fine structure with a frequency of about 1050 +/- 50 cm(-1). This is the carbonyl stretch frequency in the electronically excited state and is well-known from the vibrational structure in the CD spectra of carbonyl compounds. The positive sign of the CD in the near UV is in accordance with the octant rule and the high-resolution X-ray structure, if we assume that the NH group of cysteine 69 to which the carbonyl is hydrogen bonded is the principle perturbant. Similar absorption and CD spectra were observed in the range of 300-340 nm for the mutants E46Q and M100A at neutral pH. Protonation of the trans chromophore by lowering the pH in the dark (without photoisomerization) broadens the 307 and 318 nm CD bands in the mutant E46Q but does not significantly affect their positions or alter their sign. For the long-lived I(2) photointermediate of the mutant M100A with protonated cis chromophore, we observed that the sign of the rotational strength in the 310-320 nm range is negative (i.e., opposite to that in the dark state with trans chromophore). This suggests that the light-induced isomerization of the chromophore, which leads to breaking of the hydrogen bond with the backbone amide of C69, brings the carbonyl into a new protein environment with different asymmetry than in the unbleached protein. The observed change in sign is mainly due to this effect, but a change in chromophore twist may also contribute. Thus, the 318 nm CD signal is a sensitive marker for the environment of the chromophore carbonyl, which samples various environments and configurations during the photocycle.

Heterologous production of Halorhodospira halophila holo-photoactive yellow protein through tandem expression of the postulated biosynthetic genes.

The photoactive yellow protein (PYP) is a bacterial photoreceptor which is the structural prototype for the PAS domain superfamily of regulators and receptors. PYP is known to have a unique p-hydroxycinnamic acid chromophore, covalently attached to a cysteine. To date, it has not been shown how holo-PYP is formed in vivo. Two genes, nearby pyp, were postulated to encode the biosynthetic enzymes, but only one was previously isolated and shown to have the requisite activity. By using a dual plasmid system, one expressing the PYP from Halorhodospira halophila and the other expressing a two-gene operon, consisting of tyrosine ammonia lyase and p-hydroxycinnamic acid ligase, we are able to present evidence that a functionally active holo-PYP can be synthesized in Escherichia coli. Plasmids containing only one of the two enzymes failed to produce holoprotein. Thus, the two genes have been shown to be both necessary and sufficient for production of holoprotein, although the activating group remains unknown. This expression system not only holds great potential for mutagenesis studies but also opens new possibilities in the search for (a) signaling partner(s) of the PYP.

Discovery and characterization of electron transfer proteins in the photosynthetic bacteria.

Research on photosynthetic electron transfer closely parallels that of other electron transfer pathways and in many cases they overlap. Thus, the first bacterial cytochrome to be characterized, called cytochrome c (2), is commonly found in non-sulfur purple photosynthetic bacteria and is a close homolog of mitochondrial cytochrome c. The cytochrome bc (1) complex is an integral part of photosynthetic electron transfer yet, like cytochrome c (2), was first recognized as a respiratory component. Cytochromes c (2) mediate electron transfer between the cytochrome bc (1) complex and photosynthetic reaction centers and cytochrome a-type oxidases. Not all photosynthetic bacteria contain cytochrome c (2); instead it is thought that HiPIP, auracyanin, Halorhodospira cytochrome c551, Chlorobium cytochrome c555, and cytochrome c (8) may function in a similar manner as photosynthetic electron carriers between the cytochrome bc (1) complex and reaction centers. More often than not, the soluble or periplasmic mediators do not interact directly with the reaction center bacteriochlorophyll, but require the presence of membrane-bound intermediates: a tetraheme cytochrome c in purple bacteria and a monoheme cytochrome c in green bacteria. Cyclic electron transfer in photosynthesis requires that the redox potential of the system be delicately poised for optimum efficiency. In fact, lack of redox poise may be one of the defects in the aerobic phototrophic bacteria. Thus, large concentrations of cytochromes c (2) and c' may additionally poise the redox potential of the cyclic photosystem of purple bacteria. Other cytochromes, such as flavocytochrome c (FCSD or SoxEF) and cytochrome c551 (SoxA), may feed electrons from sulfide, sulfur, and thiosulfate into the photosynthetic pathways via the same soluble carriers as are part of the cyclic system.

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis.

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.

Protein dynamics: imidazole and 2-mercaptoethanol binding to the Rhodobacter capsulatus cytochrome c(2) mutant, glycine 95 proline.

The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics.

Crystal structures at atomic resolution reveal the novel concept of "electron-harvesting" as a role for the small tetraheme cytochrome c.

The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners.

The 1.25 A resolution structure of the diheme NapB subunit of soluble nitrate reductase reveals a novel cytochrome c fold with a stacked heme arrangement.

The diheme cytochrome NapB constitutes the small subunit of a periplasmic nitrate reductase found in a wide variety of bacterial species, including pathogens. The NapB protein is essential in transferring electrons to the large catalytic subunit NapA, which subsequently reduces nitrate to nitrite. Here we present the crystal structure of a proteolyzed form of recombinant NapB from Haemophilus influenzae, which was determined by the multiple-wavelength anomalous dispersion (MAD) method at 1.25 A resolution. This structure shows an unprecedented fold, confirming that NapB proteins belong to a new class of cytochromes. The two heme groups have nearly parallel heme planes and are stacked at van der Waals distances with an iron-to-iron distance of only 9.9 A, two structural features that are also present in the split-Soret diheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774, which is otherwise unrelated in the peptide chain folding pattern. The two propionate side chains on both heme groups are hydrogen-bonded to each other, a structural characteristic that to date also has not been reported in any other heme protein. The propionates of one of the heme groups are pulled toward the interior of the molecule due to a salt bridge and a number of hydrogen bonds between the propionates and conserved residues. We propose a hypothetical but plausible model of the NapAB complex in which the four redox centers are positioned in a virtually linear configuration which spans a distance of nearly 40 A, suggesting an efficient pathway for the transfer of electrons from NapC, the physiological electron donor of NapB, to a nitrate molecule at the catalytic site of NapA.

Evolutionary analysis by whole-genome comparisons.

A total of 37 complete genome sequences of bacteria, archaea, and eukaryotes were compared. The percentage of orthologous genes of each species contained within any of the other 36 genomes was established. In addition, the mean identity of the orthologs was calculated. Several conclusions result: (i) a greater absolute number of orthologs of a given species is found in larger species than in smaller ones; (ii) a greater percentage of the orthologous genes of smaller genomes is contained in other species than is the case for larger genomes, which corresponds to a larger proportion of essential genes; (iii) before species can be specifically related to one another in terms of gene content, it is first necessary to correct for the size of the genome; (iv) eukaryotes have a significantly smaller percentage of bacterial orthologs after correction for genome size, which is consistent with their placement in a separate domain; (v) the archaebacteria are specifically related to one another but are not significantly different in gene content from the bacteria as a whole; (vi) determination of the mean identity of all orthologs (involving hundreds of gene comparisons per genome pair) reduces the impact of errors in misidentification of orthologs and to misalignments, and thus it is far more reliable than single gene comparisons; (vii) however, there is a maximum amount of change in protein sequences of 37% mean identity, which limits the use of percentage sequence identity to the lower taxa, a result which should also be true for single gene comparisons of both proteins and rRNA; (viii) most of the species that appear to be specifically related based upon gene content also appear to be specifically related based upon the mean identity of orthologs; (ix) the genes of a majority of species considered in this study have diverged too much to allow the construction of all-encompassing evolutionary trees. However, we have shown that eight species of gram-negative bacteria, six species of gram-positive bacteria, and eight species of archaebacteria are specifically related in terms of gene content, mean identity of orthologs, or both.