Simultaneous Genome Sequencing of Prosthecochloris ethylica and Desulfuromonas acetoxidans within a Syntrophic Mixture Reveals Unique Pili and Protein Interactions.

Strains of Chloropseudomonas ethylica, 2-K, N2, and N3 are known to be composed of a syntrophic mixture of a green sulfur bacterium and a sulfur-reducing colorless component. Upon sequence analysis, the green sulfur photosynthetic bacterial component of strain N3 was dominant and was readily sequenced, but the less abundant sulfur-reducing bacterial component was apparent only when analyzed by metagenomic binning. Whole-genome comparison showed that the green bacterium belonged to the genus Prosthecochloris and apparently was a species for which there was no genome sequence on file. For comparison, we also sequenced the genome of Prosthecochloris sp. DSM 1685, which had previously been isolated from the 2-K mixture in pure culture and have shown that all three Prosthecochloris genomes belong to a new species, which we propose to be named Prosthecochloris ethylica comb. nov. Whole genomes were also sequenced for the isolated Desulfuromonas strains DSM 1675 (from strain 2-K) and DSM 1676 (from strain N2) and shown to be nearly identical to the genome found in the N3 mixture. The genome of the green sulfur bacterium contains large genes for agglutination proteins, similar to the ones proposed to be involved in larger photosynthetic consortia of Chlorochromatium aggregatum. In addition, we also identified several unique "tight adhesion (tad)" pili genes that are presumably involved in the formation of cell-cell interactions. The colorless component, on the other hand, contained a unique large multiheme cytochrome C and unique genes for e-pili (geopilin) formation, genetically clustered with a conserved ferredoxin gene, which are all expected to play an electron transfer role in the closed sulfur cycle in the syntrophic mixture. The findings from the simultaneous genome sequencing of the components of Cp. ethylica have implications for the phenomenon of direct interspecies interactions and coupled electron transfer in photosynthetic symbionts. The mechanisms for such interactions appear to be more common in the environment than originally anticipated.

Genome Sequences of Allochromatium palmeri and Allochromatium humboldtianum Expand the Allochromatium Family Tree of Purple Sulfur Photosynthetic Bacteria within the Gammaproteobacteria and Further Refine the Genus.

New genomes of two Allochromatium strains were sequenced. Whole-genome and average nucleotide identity based on BLAST (ANIb) comparisons show that Allochromatium humboldtianum is the nearest relative of Allochromatium vinosum (ANIb, 91.5%), while both Allochromatium palmeri and Thermochromatium tepidum are more distantly related (ANIb, <87%). These new sequences firmly establish the position of Allochromatium on the family tree.

Genome Sequence of the Alphaproteobacterium Blastochloris sulfoviridis DSM 729, Which Requires Reduced Sulfur as a Growth Supplement and Contains Bacteriochlorophyll b.

The genome sequence of Blastochloris sulfoviridis is 3.85 Mb with a GC content of 68%. Its nearest relative is B. tepida (average nucleotide identity [ANI], 91.5%), followed by B. viridis (ANI, 83%). According to ANI and whole-genome-based phylogenetic analysis, the nearest relatives of Blastochloris are Rhodoplanes and Rhodopseudomonas, confirming the recognition of distinct genera.

Draft Genome Sequences of Thiorhodococcus mannitoliphagus and Thiorhodococcus minor, Purple Sulfur Photosynthetic Bacteria in the Gammaproteobacterial Family Chromatiaceae.

We have determined the draft genome sequences of Thiorhodococcus mannitoliphagus and Thiorhodococcus minor for comparison with those of T. drewsii and Imhoffiella purpurea According to average nucleotide identity (ANI) and whole-genome phylogenetic comparisons, these two species are clearly distinct from the Imhoffiella species and T. drewsii.

Genome Sequence of the Acidophilic Nonsulfur Purple Photosynthetic Alphaproteobacterium Rhodovastum atsumiense, a Divergent Member of the Acetobacteraceae Family.

The genome sequence of the acidophile Rhodovastum atsumiense was determined for comparison with that of Rhodopila globiformis Both genomes are unusually large for purple bacteria (7.10 Mb and 7.25 Mb, respectively), and they have an average nucleotide identity of 72%. This value is remarkably similar to the average nucleotide identity values for Acidisphaera, Elioraea, and Paracraurococcus, all aerobic anoxygenic phototrophs.

Whole-Genome Sequence of a Unique Elioraea Species Strain Isolated from a Yellowstone National Park Hot Spring.

The genus Elioraea has only one species characterized microbiologically and two genomes sequenced. We have sequenced the genome of a unique Elioraea strain isolated from Yellowstone National Park and found it to be a distinct new species. Elioraea is suggested to be a member of the aerobic anoxygenic photosynthetic bacteria.

Draft Whole-Genome Sequence of the Green Sulfur Photosynthetic Bacterium Chlorobaculum sp. Strain 24CR, Isolated from the Carmel River.

Green sulfur bacteria are in the family Chlorobiaceae, which is composed of four distinct genera, namely, Chlorobaculum, Chlorobium, Prosthecochloris, and Chloroherpeton, with Chlorobium species being the most commonly represented in genome studies. We have now sequenced only the fourth species of Chlorobaculum, which established Chlorobaculum sp. 24CR as a separate species and should help characterize the genus.

Draft Whole-Genome Sequence of the Purple Nonsulfur Photosynthetic Bacterium Rhodopseudomonas rutila R1.

Rhodopseudomonas species are purple nonsulfur bacteria found in many environments and known for their diverse metabolic capabilities. Here, we report the genome sequence of Rhodopseudomonas rutila type strain R1 and a whole-genome nucleotide comparison of related Rhodopseudomonas palustris species, suggesting the necessity for future reevaluation of the Rhodopseudomonas species differentiation.

Draft Whole-Genome Sequence of the Purple Photosynthetic Bacterium Rhodopseudomonas palustris XCP.

Rhodopseudomonas palustris is known for its versatile metabolic capabilities and has been proposed for a wide range of innovative applications. Here, we report the genome sequence of strain XCP, as well as a whole-genome nucleotide comparison of R. palustris strains, which indicates the need for further differentiation of the known strains.

Draft Genome Sequence and Brief History of Rhodovulum sp. Strain BSW8.

Rhodovulum is a marine Gram-negative purple photosynthetic bacterial genus that is a member of the Alphaproteobacteria. Strain BSW8 is a variant that does not appear to make a polysaccharide slime capsule, and its genome sequence further contributes to the diversity of sequenced genomes belonging to this genus.

Photokinetic, biochemical and structural features of chimeric photoactive yellow protein constructs.

Of the 10 photoactive yellow protein (PYPs) that have been characterized, the two from Rhodobacter species are the only ones that have an additional intermediate spectral form in the resting state (λmax  = 375 nm), compared to the prototypical Halorhodospira halophila PYP. We have constructed three chimeric PYP proteins by replacing the first 21 residues from the N-terminus (Hyb1PYP), 10 from the ß4-ß5 loop (Hyb2PYP) and both (Hyb3PYP) in Hhal PYP with those from Rb. capsulatus PYP. The N-terminal chimera behaves both spectrally and kinetically like Hhal PYP, indicating that the Rcaps N-terminus folds against the core of Hhal PYP. A small fraction shows dimerization and slower recovery, possibly due to interaction at the N-termini. The loop chimera has a small amount of the intermediate spectral form and a photocycle that is 20 000 times slower than Hhal PYP. The third chimera, with both regions exchanged, resembles Rcaps PYP with a significant amount of intermediate spectral form (λmax  = 380 nm), but has even slower kinetics. The effects are not strictly additive in the double chimera, suggesting that what perturbs one site, affects the other as well. These chimeras suggest that the intermediate spectral form has its origins in overall protein stability and solvent exposure.

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Occurrence and sequence of Sphaeroides Heme Protein and diheme cytochrome C in purple photosynthetic bacteria in the family Rhodobacteraceae.

BACKGROUND: Sphaeroides Heme Protein (SHP) was discovered in the purple photosynthetic bacterium, Rhodobacter sphaeroides, and is the only known c-type heme protein that binds oxygen. Although initially not believed to be widespread among the photosynthetic bacteria, the gene has now been found in more than 40 species of proteobacteria and generally appears to be regulated. Rb. sphaeroides is exceptional in not having regulatory genes associated with the operon. We have thus analyzed additional purple bacteria for the SHP gene and examined the genetic context to obtain new insights into the operon, its distribution, and possible function. RESULTS: We found SHP in 9 out of 10 strains of Rb. sphaeroides and in 5 out of 10 purple photosynthetic bacterial species in the family Rhodobacteraceae. We found a remarkable similarity within the family including the lack of regulatory genes. Within the proteobacteria as a whole, SHP is part of a 3-6 gene operon that includes a membrane-spanning diheme cytochrome b and one or two diheme cytochromes c. Other genes in the operon include one of three distinct sensor kinase - response regulators, depending on species, that are likely to regulate SHP. CONCLUSIONS: SHP is not as rare as generally believed and has a role to play in the photosynthetic bacteria. Furthermore, the two companion cytochromes along with SHP are likely to function as an electron transfer pathway that results in the reduction of SHP by quinol and formation of the oxygen complex, which may function as an oxygenase. The three distinct sensors suggest at least as many separate functional roles for SHP. Two of the sensors are not well characterized, but the third is homologous to the QseC quorum sensor, which is present in a number of pathogens and typically appears to regulate genes involved in virulence.

Regulation of the Ppr histidine kinase by light-induced interactions between its photoactive yellow protein and bacteriophytochrome domains.

Ppr is a unique bacteriophytochrome that bleaches rather than forming a far-red-shifted Pfr state upon red light activation. Ppr is also unusual in that it has a blue light photoreceptor domain, PYP, which is N-terminally fused to the bacteriophytochrome domain (Bph). When both photoreceptors are activated by light, the fast phase of Bph recovery (1 min lifetime) corresponds to the formation of an intramolecular long-lived complex between the activated PYP domain and the Bph domain (lifetime of 2-3 days). Since this state is unusually long-lived as compared to other intermediates in the photocycle of both PYP and Bph, we interpret this as formation of a metastable complex between activated PYP and Bph domains that takes days to relax. In the metastable complex, the PYP domain is locked in its activated UV absorbing state and the Bph domain is in a slightly red-shifted state (from 701 to 702 nm), which is photochemically inactive to red or white light. The amount of metastable complex formed increases with the degree of prior activation of PYP, reaching a maximum of 50% when PYP is fully activated compared to 0% when no PYP is activated. The saturation of complex formation at 50% is believed to be due to light-induced heterogeneity within the Ppr dimer. UV irradiation (365 nm) of the metastable complex state photoreverses the activated PYP and the red-shifted Bph to the initial dark state within seconds. We therefore postulate that Ppr functions as a UV-red light sensor and describe the different Ppr states that can be obtained depending on the light quality. Both red and white light upregulate the autokinase activity, while it is downregulated in the dark. The physiological state of Ppr is most likely a mixture of three different states, dark, metastable complex, and red light-activated, with fractional populations whose amounts depend on the light quality of the environment and that regulate the extent of phosphorylation by the kinase.

Strong hydrogen bond between glutamic acid 46 and chromophore leads to the intermediate spectral form and excited state proton transfer in the Y42F mutant of the photoreceptor photoactive yellow protein.

In the Y42F mutant of photoactive yellow protein (PYP) the photoreceptor is in an equilibrium between two dark states, the yellow and intermediate spectral forms, absorbing at 457 and 390 nm, respectively. The nature of this equilibrium and the light-induced protonation and structural changes in the two spectral forms were characterized by transient absorption, fluorescence, FTIR, and pH indicator dye experiments. In the yellow form, the oxygen of the deprotonated p-hydroxycinnamoyl chromophore is linked by a strong low-barrier hydrogen bond to the protonated carboxyl group of Glu46 and by a weaker one to Thr50. Using FTIR, we find that the band due to the carbonyl of the protonated side chain of Glu46 is shifted from 1736 cm(-1) in wild type to 1724 cm(-1) in the yellow form of Y42F, implying a stronger hydrogen bond with the deprotonated chromophore in Y42F. The FTIR data suggest moreover that in the intermediate spectral form the chromophore is protonated and Glu46 deprotonated. Flash spectroscopy (50 ns-10 s) shows that the photocycles of the two forms are essentially the same except for a transition around 5 mus that has opposite signs in the two forms and is due to the chemical relaxation between the two dark states. The two cycles are coupled, likely by excited state proton transfer. The Y42F cycle differs from wild type by the occurrence of a new intermediate with protonated chromophore between the usual I(1) and I(2) intermediates which we call I(1)H (370 nm). Transient fluorescence measurements indicate that in I(1)H the chromophore retains the orientation it had in I(1). Transient proton uptake occurs with a time constant of 230 mus and a stoichiometry of 1. No proton uptake was associated however with the formation of the I(1)H intermediate and the relaxation of the yellow/intermediate equilibrium. These protonation changes of the chromophore thus occur intramolecularly. The chromophore-Glu46 hydrogen bond in Y42F is shorter than in wild type, since the adjacent chromophore-Y42 hydrogen bond is replaced by a longer one with Thr50. This facilitates proton transfer from Glu46 to the chromophore in the dark by lowering the barrier, leading to the protonation equilibrium and causing the rapid light-induced proton transfer which couples the cycles.

Photoactive yellow protein from the halophilic bacterium Salinibacter ruber.

A gene for photoactive yellow protein (PYP) was identified from the genome sequence of the extremely halophilic aerobic bacterium Salinibacter ruber (Sr). The sequence is distantly related to the prototypic PYP from Halorhodospira halophila (Hh) (37% identity) and contains most of the amino acid residues identified as necessary for function. However, the Sr pyp gene is not flanked by its two biosynthetic genes as in other species. To determine as to whether the Sr pyp gene encodes a functional protein, we cloned and expressed it in Escherichia coli, along with the genes for chromophore biosynthesis from Rhodobacter capsulatus. The Sr PYP has a 31-residue N-terminal extension as compared to other PYPs that appears to be important for dimerization; however, truncation of these extra residues did not change the spectral and photokinetic properties. Sr PYP has an absorption maximum at 431 nm, which is at shorter wavelengths than the prototypical Hh PYP (at 446 nm). It is also photoactive, being reversibly bleached by either blue or white light. The kinetics of dark recovery is slower than any of the PYPs reported to date (4.27 x 10(-4) s(-1) at pH 7.5). Sr PYP appears to have a normal photocycle with the I1 and I2 intermediates. The presence of the I2' intermediate is also inferred on the basis of the effects of temperature and alchohol on recovery. Sr PYP has an intermediate spectral form in equilibrium with the 431 nm form, similar to R. capsulatus PYP and the Y42F mutant of Hh PYP. Increasing ionic strength stabilizes the 431 nm form at the expense of the intermediate spectral form, and the kinetics of recovery is accelerated 6.4-fold between 0 and 3.5 M salt. This is observed with ions from both the chaotropic and the kosmotropic series. Ionic strength also stabilizes PYP against thermal denaturation, as the melting temperature is increased from 74 degrees C in buffer alone to 92 degrees C in 2 M KCl. Sr accumulates KCl in the cytoplasm, like Halobacterium, to balance osmotic pressure and has very acidic proteins. We thus believe that Sr PYP is an example of a halophilic protein that requires KCl to electrostatically screen the excess negative charge and stabilize the tertiary structure.

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination.

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.

Structural role of tyrosine 98 in photoactive yellow protein: effects on fluorescence, gateway, and photocycle recovery.

We have recently shown that the Y98Q mutant of PYP has a major effect on the photocycle kinetics ( approximately 40 times slower recovery). We have now determined the crystal structure of Y98Q at 2.2 A resolution to reveal the role of residue Y98 in the PYP photocycle. Although the overall structure is very similar to that of WT, we observed two major effects of the mutation. One obvious consequence is a conformational change of the beta4-beta5 loop, which includes a repositioning of residue M100. It had previously been shown that the photocycle is slowed by as much as 3 orders of magnitude when residue M100 is substituted or when the conformation is altered as in Rhodocista centenaria PYP. To investigate whether the altered photocycle of Y98Q is due to this repositioning of M100 or is caused by an effect unrelated to M100, we determined the dark recovery kinetics of the Y98Q/M100A mutant. We find the recovery kinetics to be very similar to the M100A single mutant kinetics and therefore conclude that the slower recovery kinetics in Y98Q are most likely due to repositioning of M100. In addition, we find that other substitutions at position 98 (Y98W, Y98L, and Y98A) have differing effects on the photocycle recovery, presumably due to a variable distortion of the beta4-beta5 loop. The second effect of the Y98Q mutation is a repositioning of R52, which is thought to interact with Y98 in WT PYP and now forms new interactions with residues Q99 and Q56. To determine the role of R52, we also characterized an R52A/M100A double mutant and found that the effects on the recovery kinetics ( approximately 2000 slower recovery than WT) are due to unrelated events in the photocycle. Since the Y98Q/M100A recovery kinetics are more similar to those of M100 than R52A/M100A, we conclude that the repositioning of R52, caused by the Y98Q mutation, does not affect the dark state recovery. In addition, it has been proposed that Y98 and P68 are "gateway residues" between which the chromophore must pass during isomerization. We tested the recovery kinetics of mutant P68A and found that, although the gateway may be important for photocycle initiation, its role in recovery to the ground state is minimal.

Photocycle and photoreversal of photoactive yellow protein at alkaline pH: kinetics, intermediates, and equilibria.

Since the habitat of Halorhodospira halophila is distinctly alkaline, we investigated the kinetics and intermediates of the photocycle and photoreversal of the photoreceptor photoactive yellow protein (PYP) from pH 8 to 11. SVD analysis of the transient absorption time traces in a broad wavelength range (330-510 nm) shows the presence of three spectrally distinct species (I1, I1', and I2') at pH 10. The spectrum of I1' was obtained in two different ways. The maximal absorption is at 425 nm. I1' probably has a deprotonated chromophore and may be regarded as the alkaline form of I2'. At pH 10, the I1 intermediate decays in approximately 330 micros in part to I1' before I1 and I1' decay further to I2' in approximately 1 ms. From the rise of I2' (approximately 1 ms) to the end of the photocycle, the three intermediates (I1, I1', and I2') remain in equilibrium and decay together to P in approximately 830 ms. Assuming that the spectra of I1, I1', and I2' are pH-independent, their time courses were determined. On the millisecond to second time scale, they are in a pH-dependent equilibrium with a pKa of approximately 9.9. With an increase in pH, the I1 and I1' populations increase at the expense of the amount of I2'. The apparent rate constant for the recovery of P slows with an increase in pH with a pKa of approximately 9.7. The equal pH dependence of this rate and the equilibrium concentrations follows, if we assume that the equilibration rates between the intermediates are much faster than the recovery rate and that the recovery occurs from I2'. The pKa of approximately 9.9 is assigned to the deprotonation of the phenol of the surface-exposed chromophore in the I1'-I2' equilibrium. The I1-I1' equilibrium is pH-independent. Photoreversal experiments at pH 10 with the second flash at 355 nm indicate the presence of only one I2-like intermediate, which we assign on the basis of its lambda(max) value to I2'. After the rapid unresolved photoisomerization to I2'(trans), the reversal pathway back to P involves two sequential steps (60 micros and 3 ms). The amplitude spectra show that I1'(trans) and I1(trans) intermediates participate in this reversal.