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Halorhodospira halophila - Biology


Halorhodospira halophila (formerly Ectothiorhodospira halophila) is an extremely halophilic purple bacterium that was formally a member of the Ectothiorhodospira genus until recently reclassified. Phylogenetically, Halorhodospira halophila is associated within the gamma subdivision of the phylum Proteobacteria and is known to be phototrophic and Gram-negative. This importantly shows that harsh environments once thought to be exclusive to archaea actually contain bacteria as well.


Substrate-binding region of ABC-type glycine betaine transport system

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Light-harvesting protein B800/850/890 alpha-3 chain

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Halorhodospira Halophila

3.2 The Photocycle of the Photoactive Yellow Protein

The PYP, which was first discovered in the cytoplasm of Halorhodospira halophila bacterium, is a cytoplasmic blue light photoreceptor initiates the negative phototaxis pathway. Upon photoirradiation, the chromophore of PYP undergoes a fully reversible photocycle starting from a dark-state pG and then going through two early spectroscopic intermediates I0 and I0 ‡ that may be equivalent to IT and ICP, respectively. Following the initial relaxation, wt-PYP decays quickly to a red-shifted intermediate pR. However, the precise mechanism for generating pR is still unclear. Ihee et al. ( Jung et al., 2013 Kim et al., 2012 ) proposed that IT may simultaneously decay to the two intermediates pR1 and pR2 (via ICP) through a parallel isomerization reaction, while Zimányi et al. ( Khoroshyy et al., 2013 ) proposed different mechanism that pR2 is produced after pR1 via a sequential reaction. In both mechanisms the intermediate pR2 undergoes a protonation reaction to capture one proton from the Glu46 residue, leading to a blue-shifted intermediate pB′ and an energetically unstable, charged Glu46 − . The Glu46 − triggers a large conformational change in the protein, relaxes to a putative signaling state pB which forms a new blue-shifted intermediate. Ihee et al. ( Kim et al., 2012 ) also published another possibility the pB may be reached directly from pR1 because they exhibit similarities in the chromophore orientation and the surrounding hydrogen-bonding networks. Eventually, the photocycle is completed via a series of deprotonation and reisomerization processes. A structural recovery of PYP is achieved through a new intermediate pG′ with a deprotonated chromophore that facilitates the occurrence of reisomerization. Identify all possible intermediates in the photocycle of PYP is significant challenges for both experimental and theoretical investigations. Since there are so many debated issues, such as the parallel versus sequential kinetic pathways, the assignment of various intermediates, and the identity of the donor and acceptor for proton transfer in the multistep protonation–deprotonation reaction, Chen and Fang et al. ( Wei et al., 2014 ) carry out a comprehensive computational study to address the overall process using a combined QM/MM approach, at the level of theory of CASPT2/CASSCF/AMBER. In the computation of the constrained MEPs along the physically motivated reaction coordinates modeling all possible photoisomerization and proton transfer processes, the pCA chromophore, and its adjacent residues (complete or partial) as well as the crystal water molecules are included in QM subsystem, while the remaining residues, water molecules, and counterions were treated using MM. Different QM/MM partitions were adopted to account for various steps in the photocycle of PYP. The first step of photoisomerization is described with QM1, which includes the pCA chromophore and a portion of the Glu46 and Cys69 residues. The latter isomerization steps ICP → pR2 and pR1 → pR2, as well as subsequent processes, which involve two typical structural deformations via the simultaneous torsion along two nonadjacent or adjacent bonds, are described by QM2 with whole Cys69 residue included. To compute the MEPs of the protonation–deprotonation steps, additional one and three crystal water molecules have been added to the QM3 and QM4 subsystems, respectively. For the QM3, part of Tyr42 was also added to account for its role of proton transfer relay.

Their study provides a comprehensive picture of the overall photocycle of the isomerization and protonation reactions upon the photoexcitation of the wild-type PYP. As illustrated in Scheme 3 , after photoirradiation to S1( 1 ππ*) state, the pG isomerizes along b bond through one-bond flip (OBF) to CI (S0/S1), followed by “hula twist” (HT), “bicycle pedal”(BP), and OBF isomerization reactions to form cis isomers pR1, ICP, and ICT, respectively. The pB could be achieved by either protonation of pR1 or ICP → pB transition. Starting from ICP, a barrier of 16.2 kcal/mol bicycle pedal isomerization needs to over to form an intermediate pR2, with a fast protonation reaction (6.1 kcal/mol) to get pB′. There is another high barrier of 28.4 kcal/mol that needs to surmount through bicycle pedal isomerization and a reverse OBF isomerization, which leads to a hydrophobic–hydrophilic transformation and the formation of pB. After that, the exposed COO − group of Glu46 attracts water solvent into the binding pocket and triggers the fast deprotonation reaction of pCA. The photocycle is finally completed by the recovery of the phenoxy ring repelling water molecules out of the binding pocket through an OBF isomerization with a large barrier of 36.6 kcal/mol. Their studies unveil that the parallel mechanism through pR1 → pB transition is a favorable channel but coexists with the sequential model via the pR1 → pR2 transformation.

Scheme 3 . Mechanistic illustration of the overall PYP photocycle: the protonation–deprotonation and isomerization reactions of the hula twist (HT), bicycle pedal (BP), and one-bond flip (OBF) are shown in blue (light gray in the print version) along the special one or two bonds and the related barriers (kcal/mol) are also highlighted in red (light gray in the print version).

From Wei et al. (2014) , reproduced by permission of the PCCP Owner Societies.


Genome sequencing and annotation

Genome project history

This organism was selected for sequencing to better understand its halophilic adaptations, its unusual sulfur metabolism, its photosynthetic pathways, and to provide a framework for better understanding signaling pathways for photoactive yellow protein. The complete genome sequence has been deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). Table 2 presents the project information and its association with MIGS version 2.0 compliance [25].

Growth conditions and DNA isolation

H. halophila SL1 strain DSM 44 T was obtained from Deutsche Sammlung vor Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and were grown in DSMZ 253 medium. The cells were grown anaerobically and photosynthetically by placing them in 20 ml glass culture tubes completely filled with growth medium and sealed with screw caps. The tubes were kept at 42ºC in a water bath and illuminated with 70 W tungsten light bulbs. Chromosomal DNA was purified from the resulting cell cultures using the CTAB procedure.

Genome sequencing and assembly

The random shotgun method was used in sequencing the genome of H. halophila SL1. Large (40 kb), median (8 kb) and small (3 kb) insert random sequencing libraries were sequenced for this genome project with an average success rate of 88% and average high-quality read lengths of 750 nucleotides. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher (unpublished, C. Han) or by transposon bombing of bridging clones (EZ-Tn5 <P6Kyori/KAN-2> Tnp Transposome kit, Epicentre Biotechnologies). Gaps between contigs were closed by editing, custom primer walks or PCR amplification. The completed genome sequence of H. halophila SL1 contains 36,035 reads, achieving an average of 12-fold sequence coverage per base with error rate less than 1 in 100,000.

Genome annotation

Identification of putative protein-encoding genes and initial automated annotation of the genome was performed by the Oak Ridge National Laboratory genome annotation pipeline. Additional gene prediction analysis and functional annotation was performed within the IMG platform [41].


Side-chain specific isotopic labeling of proteins for infrared structural biology: the case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.


Interaction of a photochromic UV sensor protein Rc-PYP with PYP-binding protein

Photoactive yellow protein (PYP) from Halorhodospira halophila is one of typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were studied. It was found that UV light-induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-II). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-I) is formed transiently, which produced Complex-II via a second-order reaction. Any other intermediates, including those from pBL do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-II dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor, of which signaling process is similar to that of other light sensor proteins in the visible light region. The photochromic heterogeneous intermolecular interactions formed between PYP and PBP can be used as a novel and useful tool in optogenetics.


Structure and mechanistic features of the prokaryotic minimal RNase P

Endonucleolytic removal of 5’-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives) both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5’-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.


Deuterium isotope effects in the photocycle transitions of the photoactive yellow protein

The Photoactive Yellow Protein (PYP) from Halorhodospira halophila (formerly Ectothiorhodospira halophila) is increasingly used as a model system. As such, a thorough understanding of the photocycle of PYP is essential. In this study we have combined information from pOH- (or pH-) dependence and (kinetic) deuterium isotope effects to elaborate on existing photocycle models. For several characteristics of PYP we were able to make a distinction between pH- and pOH-dependence, a nontrivial distinction when comparing data from samples dissolved in H(2)O and D(2)O. It turns out that most characteristics of PYP are pOH-dependent. We confirmed the existence of a pB' intermediate in the pR to pB transition of the photocycle. In addition, we were able to show that the pR to pB' transition is reversible, which explains the previously observed biexponential character of the pR-to-pB photocycle step. Also, the absorption spectrum of pB' is slightly red-shifted with respect to pB. The recovery of the pG state is accompanied by an inverse kinetic deuterium isotope effect. Our interpretation of this is that before the chromophore can be isomerized, it is deprotonated by a hydroxide ion from solution. From this we propose a new photocycle intermediate, pB(deprot), from which pG is recovered and which is in equilibrium with pB. This is supported in our data through the combination of the observed pOH and pH dependence, together with the kinetic deuterium isotope effect.

Figures

Spectral deuterium isotope effect. In…

Spectral deuterium isotope effect. In ( A ), the fitted spectra of the…

KDIE obtained with the simple…

KDIE obtained with the simple photocycle model. In ( A ) the photocycle…

Comparison between the simple and…

Comparison between the simple and the complex model. Data recorded at 360 (…

KDIE obtained with the complex…

KDIE obtained with the complex model. In ( A ) the photocycle model…


Introduction

All halophilic organisms face the risk of cellular dehydration caused by the high osmotic activity of saline environments, and require osmoprotection strategies to survive. Since saline oceans, saline lakes, inland seas, and saline groundwater constitute

97% of all water on earth 1 , and salt deposits underlay roughly one quarter of the land on earth 2 , saline and hypersaline environments are of great ecological significance. In addition, salinity has been identified as a major determinant for microbial community composition 3 . Therefore, halophilic adaptations are of general biological interest. Halophilic microorganisms manage to thrive in saline and even hypersaline environments by increasing the osmotic activity of their cytoplasm to match that of the environment. Two types of osmoadaptive strategies are employed by halophiles and extreme halophiles 4,5,6,7 : either the accumulation of molar concentrations of KCl or the accumulation of organic compounds such as glycine betaine compatible solutes in their cytoplasm 8,9,10,11 . Which of these two main osmoprotection strategies an organism utilizes has profound implications for its ecology, physiology, biochemistry, and evolutionary history.

Organic osmolytes do not interfere with the functional properties of cytoplasmic components 10,12,13,14 , and are thus referred to as compatible solutes. For organisms utilizing this osmoprotection strategy it therefore suffices to accumulate compatible solutes either by uptake from the extracellular medium or by biosynthesis. However, the biosynthesis of molar concentrations of organic osmoprotectants represents a large investment in terms of ATP, reducing equivalents, and carbon 5 . In contrast, high concentrations of KCl are generally toxic to various cytoplasmic enzymes 15 . In organisms using KCl as their main osmoprotectant the profile of isoelectric points of the entire proteome is shifted to acidic values 16,17,18,19,20,21,22,23 . Such proteome acidity is believed to allow enzyme function to proceed in the saline cytoplasm 19,20,21,24,25 , but is thought to require the organism to permanently maintain high levels of cytoplasmic KCl. Once an organism has evolved an acidic proteome, a key advantage is that KCl is a bioenergetically inexpensive osmoprotectant. Haloarchaea such as Halobacterium salinarum rely almost entirely on KCl, while many halophilic Bacteria predominantly use glycine betaine 26,27 . Almost all halophiles and extreme halophiles for which osmoprotectant levels have been reported exclusively use either KCl or compatible solutes as their main osmoprotectants 4,5,6 . This overarching view that has guided thinking regarding the dual nature of osmoprotection strategies has been challenged by recent experimental results 28,29 , which prompted us to perform the studies reported here.

Most knowledge regarding halophilic osmoprotection strategies has been obtained through extensive studies of a very small number of organisms, particularly the Archaeon Halobacterium salinarum 17 and Salinibacter ruber (Bacteroidetes) 22,30 . Therefore, we aimed to examine the osmoprotection strategy of extreme halophiles from other bacterial phyla, and selected two closely related 31 extremely halophilic purple photosynthetic Proteobacteria: Halorhodospira halophila 32 and Halorhodospira halochloris 33 . While H. halochloris has been shown to utilize glycine betaine as its main osmoprotectant 34 , we found that H. halophila predominantly accumulates molar concentrations of KCl 28 . This result implies a relatively recent and dramatic evolutionary change in osmoprotection strategy. Unexpectedly, we found that at lower salinity (approximately that of sea water) H. halophila retains an acidic proteome but does not accumulate cytoplasmic KCl beyond levels found in E. coli 28 . This observation does not follow the expectation that acidic proteomes in halophiles require high levels of KCl to be functional. In addition, further studies revealed that many Halobacteriales are not limited to the use of KCl as their osmoprotectant but utilize organic osmoprotectants 29 . Taken together, these observations imply an unexpected diversity in osmoprotection mechanisms. While most extreme halophiles are believed to dedicate their osmoprotection strategy either to the use of KCl or organic osmoprotectants, here we report the existence of an “omoprotection switch” in H. halophila, allowing this organism to switch between the KCl and organic osmoprotectant strategies depending on environmental conditions.


Discussion

HiPIP II Is the Physiological Electron Donor to the Photosynthetic RC in H. halophila. As detailed above, a significant fraction of HiPIP present in the periplasm of H. halophila cosediments with the membrane during preparation of chromatophores. This HiPIP has been identified as HiPIP II. The facts that this HiPIP (i) can be photooxidized in immobilized samples and (ii) is bound to the membranes in a well defined conformation in photo-oxidized or chemically oxidized samples, demonstrate that part if not all of HiPIP II forms a tight and electron transfer competent complex with the RC. The kinetics of light-induced absorption changes corroborate the existence of this complex and show that the HiPIP to P + electron transfer is mediated by a cytochrome subunit. A docking site for HiPIP II on this subunit is in line with the observation that crude extracts of HiPIP II containing a cytochrome component retain the g1 = 2.10 signal. The kinetic data yield a t 1/2 of ≤11 μs for electron donation from HiPIP to the cytochrome subunit.

The results therefore demonstrate that HiPIP II is the electron carrier re-reducing the RC in H. halophila by means of a cytochrome subunit (at present there is no evidence for involvement of another electron carrier). Both the optical and the EPR experiments show a substoichiometric oxidation of the RC-bound HiPIP. A major part of this “missing” HiPIP photooxidation can be attributed to the equilibrium constant between P + and HiPIP. The observed strong deceleration of the donation reaction from the cytochrome subunit to P + and the decrease of the extent of P + oxidation increasing the ambient redox potential provides evidence for a significantly lower ΔE m between P + , heme, and HiPIP in the coupled system (at ambient potentials where the high potential pair of hemes is reduced) than expected from equilibrium titrations. Effects of electrostatic interactions on kinetic reactions in RCs have been detailed in ref. 38.

HiPIP II from H. halophila Shows Strongly Variable EPR and Redox Properties. The RC-bound HiPIP II has EPR spectra significantly different from those published and confirmed in this work for the soluble HiPIP II. Ironically, the shift of g 1 from 2.10 in the RC-bound state to 2.14 in the purified protein is documented in two previous articles (26, 31) but has previously been overlooked. Among all HiPIPs studied in the past, the soluble HiPIP II from H. halophila stands out by its spectral simplicity. Although HiPIPs typically show “complicated” EPR spectra probably arising from the superposition of two or more paramagnetic centers, the spectrum of soluble H. halophila HiPIP II can be simulated by a single paramagnetic species with g 1 at 2.14, g 2 at 2.034, and g 3 at 2.024 (33). NMR data suggest that the “simplicity” of the HiPIP II EPR species arises from a single positioning of the mixed valence pair in the oxidized cluster (39), in contrast to two or more permutations of this pair among the four iron atoms of the cubane in other HiPIPs. As shown above, this single paramagnetic species in the purified protein differs from the single paramagnetic species in the RC-bound HiPIP II. Interaction of HiPIP II with the cytochrome subunit thus most probably turns a different mixed valence pair distribution into the lowest energy state, resulting in distinguishable paramagnetic species in soluble and RC-bound HiPIP. An attribution of g-tensor to molecular axes has been determined for HiPIP II based on electron-nuclear double resonance (ENDOR) results (33). The likely change of mixed valence localization, however, unfortunately precludes a structural interpretation of the g-tensor orientations determined in our work.

In summary, although the molecular bases for the observed E m and paramagnetic variabilities are not clear at present, they nevertheless (i) provide an excellent tool for analyzing the interactions of HiPIP II with its redox partners and (ii) permit us to understand the functional energetics of the H. halophila electron transfer chain (see next section).

The Solution to the “Redox Enigma.” Previously, the low E m values of the two iso-HiPIPs from H. halophila (+50 mV and +110 mV determined at low salt and in the isolated state) (40) were taken as evidence against their implication in photosynthetic electron transfer. The Em values of P/P + in the RC and the Rieske center in the bc 1 complex were tacitly assumed to be in the typical range for purple bacteria (i.e., +400 to +500 mV for P/P + and +250 to +340 mV for Rieske). In this work, the E m of HiPIP II at physiological salt concentrations and in the complexed state was determined to be +140 ± 20 mV. In our studies of H. halophila, we measured the redox potentials of P/P + and the hemes present in the cytochrome subunit of the photosynthetic RC, as well as the redox potential of the Rieske cluster of the bc 1 complex (37). The Em values of P/P + and of the Rieske center in H. halophila were found to be +270 mV and +120 ± 20 mV, respectively, at physiological salt conditions. Electron transfer from the Qo-site of the bc 1 complex to the photosynthetic RC thus occurs in the range between +100 mV and +300 mV, i.e., >100 mV below the values encountered in other purple bacterial systems. Moreover, the potential of HiPIP II in physiological salt conditions is at least 70 mV more positive than that determined previously under standard conditions. These two corrections to previously assumed potentials render HiPIP II well suited for shuttling electrons between the bc 1 complex and the RC. So far, none of our results indicate an implication of HiPIP I in photosynthetic electron transfer. The functional role of HiPIP I thus remains to be elucidated.

The HiPIP Electron Shuttle in Proteobacteria: Merely a Substitute for Soluble Cytochromes? Because the first reports demonstrated that HiPIPs can play a functional role equivalent to that of soluble cytochrome c 2 (10, 11), a number of studies have (i) shown the widespread occurrence of HiPIPs in proteobacterial electron transfer chains (26) and (ii) stimulated the characterization of the kinetic parameters of the HiPIP/RC electron donation reaction (10, 41, 42). The bulk of the data obtained to date has been interpreted with the HiPIPs functioning like soluble cytochromes c 2 (with slightly slower electron transfer rates) and are therefore alternatives to the soluble cytochromes just like the small copper proteins in some proteobacteria or in oxygenic photosynthesis. However, evidence is accumulating indicating that the electron transfer mediated by HiPIPs may be substantially different from that of cytochome c 2.

Site-directed mutagenesis experiments on the Rubrivivax gelatinosus tetraheme subunit (18, 20, 21, 23) have already indicated that the docking of HiPIP to the cytochrome subunit involved hydrophobic rather than electrostatic interactions as is the case for cytochrome c 2.

The interaction between HiPIP and its membranous partner is strong. HiPIP II is routinely retained during preparation of membranes, and specific efforts must be made to fully deplete the membrane fraction of HiPIP molecules. This tight binding is not restricted to the case of HiPIP II in H. halophila. We have in fact observed copurification of HiPIPs with membranes in all of the proteobacterial species that we have studied so far (22, 37). A significant affinity toward the membrane-integral reaction partners thus seems to be a common feature of proteobacterial HiPIPs. This behavior contrasts with the situation encountered for cytochrome c 2, which, although frequently retained in sealed vesicles during the preparation of chromatophores, does not show a particularly strong tendency to copurify with the membrane fraction. Our finding of strong association of HiPIP with the RC in all proteobacteria studied in fact poses a kinetic problem, because a tight complex is expected to hinder rapid exchange of a photooxidized HiPIP for another reduced HiPIP. Further experiments will be required to solve this electron transfer riddle.

A third particularity of HiPIPs, observed also with HiPIP II from H. halophila, consists in their strong tendency to multimerize in solution (37). It seems unavoidable to us that the high local concentration of HiPIP in the periplasm must increase its tendency to form multimers. Either such multimers have physiological relevance during electron transfer or another unspecified protein keeps the HiPIP from multimerizing in whole cells.

In summary, the results reported in this work together with previously published data indicate that the detailed functional mechanism of HiPIP electron shuttling may substantially deviate from that used by cytochrome c 2.