Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit.

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.

Quantum coherent energy transport in the Fenna-Matthews-Olson complex at low temperature.

In the primary step of natural light harvesting, the solar photon energy is captured in a photoexcited electron-hole pair, or an exciton, in chlorophyll. Its conversion to chemical potential occurs in the special pair reaction center, which is reached by downhill ultrafast excited-state energy transport through a network of chromophores. Being inherently quantum, transport could in principle occur via a matter wave, with vast implications for efficiency. How long a matter wave remains coherent is determined by the intensity by which the exciton is disturbed by the noisy biological environment. The stronger this is, the stronger the electronic coupling between chromophores must be to overcome the fluctuations and phase shifts. The current consensus is that under physiological conditions, quantum coherence vanishes on the 10-fs time scale, rendering it irrelevant for the observed picosecond transfer. Yet, at low-enough temperature, quantum coherence should in principle be present. Here, we reveal the onset of longer-lived electronic coherence at extremely low temperatures of ∼20 K. Using two-dimensional electronic spectroscopy, we determine the exciton coherence times in the Fenna-Matthew-Olson complex over an extensive temperature range. At 20 K, coherence persists out to 200 fs (close to the antenna) and marginally up to 500 fs at the reaction center. It decays markedly faster with modest increases in temperature to become irrelevant above 150 K. At low temperature, the fragile electronic coherence can be separated from the robust vibrational coherence, using a rigorous theoretical analysis. We believe that by this generic principle, light harvesting becomes robust against otherwise fragile quantum effects.

Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

Light harvesting in purple bacteria does not rely on resonance fine-tuning in peripheral antenna complexes.

The ring-like peripheral light-harvesting complex 2 (LH2) expressed by many phototrophic purple bacteria is a popular model system in biological light-harvesting research due to its robustness, small size, and known crystal structure. Furthermore, the availability of structural variants with distinct electronic structures and optical properties has made this group of light harvesters an attractive testing ground for studies of structure-function relationships in biological systems. LH2 is one of several pigment-protein complexes for which a link between functionality and effects such as excitonic coherence and vibronic coupling has been proposed. While a direct connection has not yet been demonstrated, many such interactions are highly sensitive to resonance conditions, and a dependence of intra-complex dynamics on detailed electronic structure might be expected. To gauge the sensitivity of energy-level structure and relaxation dynamics to naturally occurring structural changes, we compare the photo-induced dynamics in two structurally distinct LH2 variants. Using polarization-controlled 2D electronic spectroscopy at cryogenic temperatures, we directly access information on dynamic and static disorder in the complexes. The simultaneous optimal spectral and temporal resolution of these experiments further allows us to characterize the ultrafast energy relaxation, including exciton transport within the complexes. Despite the variations in PPC molecular structure manifesting as clear differences in electronic structure and disorder, the energy-transport and-relaxation dynamics remain remarkably similar. This indicates that the light-harvesting functionality of purple bacteria within a single LH2 complex is highly robust to structural perturbations and likely does not rely on finely tuned electronic- or electron-vibrational resonance conditions.

Non-canonical Staphylococcus aureus pathogenicity island repression.

Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

Quantum chemical elucidation of a sevenfold symmetric bacterial antenna complex.

The light-harvesting complex 2 (LH2) of purple bacteria is one of the most studied photosynthetic antenna complexes. Its symmetric structure and ring-like bacteriochlorophyll arrangement make it an ideal system for theoreticians and spectroscopists. LH2 complexes from most bacterial species are thought to have eightfold or ninefold symmetry, but recently a sevenfold symmetric LH2 structure from the bacterium Mch. purpuratum was solved by Cryo-Electron microscopy. This LH2 also possesses unique near-infrared absorption and circular dichroism (CD) spectral properties. Here we use an atomistic strategy to elucidate the spectral properties of Mch. purpuratum LH2 and understand the differences with the most commonly studied LH2 from Rbl. acidophilus. Our strategy exploits a combination of molecular dynamics simulations, multiscale polarizable quantum mechanics/molecular mechanics calculations, and lineshape simulations. Our calculations reveal that the spectral properties of LH2 complexes are tuned by site energies and exciton couplings, which in turn depend on the structural fluctuations of the bacteriochlorophylls. Our strategy proves effective in reproducing the absorption and CD spectra of the two LH2 complexes, and in uncovering the origin of their differences. This work proves that it is possible to obtain insight into the spectral tuning strategies of purple bacteria by quantitatively simulating the spectral properties of their antenna complexes.

Excited-State Dynamics of All the Mono-cis and the Major Di-cis Isomers of ß-Apo-8'-Carotenal as Revealed by Femtosecond Time-Resolved Transient Absorption Spectroscopy.

Cis isomers of carotenoids play important roles in light harvesting and photoprotection in photosynthetic bacteria, such as the reaction center in purple bacteria and the photosynthetic apparatus in cyanobacteria. Carotenoids containing carbonyl groups are involved in efficient energy transfer to chlorophyll in light-harvesting complexes, and their intramolecular charge-transfer (ICT) excited states are known to be important for this process. Previous studies, using ultrafast laser spectroscopy, have focused on the central-cis isomer of carbonyl-containing carotenoids, revealing that the ICT excited state is stabilized in polar environments. However, the relationship between the cis isomer structure and the ICT excited state has remained unresolved. In this study, we performed steady-state absorption and femtosecond time-resolved absorption spectroscopy on nine geometric isomers (7-cis, 9-cis, 13-cis, 15-cis, 13'-cis, 9,13'-cis, 9,13-cis, 13,13'-cis, and all-trans) of ß-apo-8'-carotenal, whose structures are well-defined, and discovered correlations between the decay rate constant of the S1 excited state and the S0-S1 energy gap, as well as between the position of the cis-bend and the degree of stabilization of the ICT excited state. Our results demonstrate that the ICT excited state is stabilized in polar environments in cis isomers of carbonyl-containing carotenoids and suggest that the position of the cis-bend plays an important role in the stabilization of the excited state.

Intraband dynamics and exciton trapping in the LH2 complex of Rhodopseudomonas acidophila.

Over the last several decades, the light-harvesting protein complexes of purple bacteria have been among the most popular model systems for energy transport in excitonic systems in the weak and intermediate intermolecular coupling regime. Despite this extensive body of scientific work, significant questions regarding the excitonic states and the photo-induced dynamics remain. Here, we address the low-temperature electronic structure and excitation dynamics in the light-harvesting complex 2 of Rhodopseudomonas acidophila by two-dimensional electronic spectroscopy. We find that, although at cryogenic temperature energy relaxation is very rapid, exciton mobility is limited over a significant range of excitation energies. This points to the presence of a sub-200 fs, spatially local energy-relaxation mechanism and suggests that local trapping might contribute substantially more in cryogenic experiments than under physiological conditions where the thermal energy is comparable to or larger than the static disorder.

A comparative look at structural variation among RC-LH1 'Core' complexes present in anoxygenic phototrophic bacteria.

All purple photosynthetic bacteria contain RC-LH1 'Core' complexes. The structure of this complex from Rhodobacter sphaeroides, Rhodopseudomonas palustris and Thermochromatium tepidum has been solved using X-ray crystallography. Recently, the application of single particle cryo-EM has revolutionised structural biology and the structure of the RC-LH1 'Core' complex from Blastochloris viridis has been solved using this technique, as well as the complex from the non-purple Chloroflexi species, Roseiflexus castenholzii. It is apparent that these structures are variations on a theme, although with a greater degree of structural diversity within them than previously thought. Furthermore, it has recently been discovered that the only phototrophic representative from the phylum Gemmatimonadetes, Gemmatimonas phototrophica, also contains a RC-LH1 'Core' complex. At present only a low-resolution EM-projection map exists but this shows that the Gemmatimonas phototrophica complex contains a double LH1 ring. This short review compares these different structures and looks at the functional significance of these variations from two main standpoints: energy transfer and quinone exchange.

Revisiting high-resolution crystal structure of Phormidium rubidum phycocyanin.

The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in ß-subunit of pr-PC), in which all residues except one (ß145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC-MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.

Nature does not rely on long-lived electronic quantum coherence for photosynthetic energy transfer.

During the first steps of photosynthesis, the energy of impinging solar photons is transformed into electronic excitation energy of the light-harvesting biomolecular complexes. The subsequent energy transfer to the reaction center is commonly rationalized in terms of excitons moving on a grid of biomolecular chromophores on typical timescales [Formula: see text]100 fs. Today's understanding of the energy transfer includes the fact that the excitons are delocalized over a few neighboring sites, but the role of quantum coherence is considered as irrelevant for the transfer dynamics because it typically decays within a few tens of femtoseconds. This orthodox picture of incoherent energy transfer between clusters of a few pigments sharing delocalized excitons has been challenged by ultrafast optical spectroscopy experiments with the Fenna-Matthews-Olson protein, in which interference oscillatory signals up to 1.5 ps were reported and interpreted as direct evidence of exceptionally long-lived electronic quantum coherence. Here, we show that the optical 2D photon echo spectra of this complex at ambient temperature in aqueous solution do not provide evidence of any long-lived electronic quantum coherence, but confirm the orthodox view of rapidly decaying electronic quantum coherence on a timescale of 60 fs. Our results can be considered as generic and give no hint that electronic quantum coherence plays any biofunctional role in real photoactive biomolecular complexes. Because in this structurally well-defined protein the distances between bacteriochlorophylls are comparable to those of other light-harvesting complexes, we anticipate that this finding is general and directly applies to even larger photoactive biomolecular complexes.

Assessing density functional theory in real-time and real-space as a tool for studying bacteriochlorophylls and the light-harvesting complex 2.

We use real-time density functional theory on a real-space grid to calculate electronic excitations of bacteriochlorophyll chromophores of the light-harvesting complex 2 (LH2). Comparison with Gaussian basis set calculations allows us to assess the numerical trust range for computing electron dynamics in coupled chromophores with both types of techniques. Tuned range-separated hybrid calculations for one bacteriochlorophyll as well as two coupled ones are used as a reference against which we compare results from the adiabatic time-dependent local density approximation (TDLDA). The tuned range-separated hybrid calculations lead to a qualitatively correct description of the electronic excitations and couplings. They allow us to identify spurious charge-transfer excitations that are obtained with the TDLDA. When we take into account the environment that the LH2 protein complex forms for the bacteriochlorophylls, we find that it substantially shifts the energy of the spurious charge-transfer excitations, restoring a qualitatively correct electronic coupling of the dominant excitations also for TDLDA.

Ultrafast energy relaxation in single light-harvesting complexes.

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

The role of charge-transfer states in the spectral tuning of antenna complexes of purple bacteria.

The LH2 antenna complexes of purple bacteria occur, depending on light conditions, in various different spectroscopic forms, with a similar structure but different absorption spectra. The differences are related to point changes in the primary amino acid sequence, but the molecular-level relationship between these changes and the resulting spectrum is still not well understood. We undertook a systematic quantum chemical analysis of all the main factors that contribute to the exciton structure, looking at how the environment modulates site energies and couplings in the B800-850 and B800-820 spectroscopic forms of LH2. A multiscale approach combining quantum chemistry and an atomistic classical embedding has been used where mutual polarization effects between the two parts are taken into account. We find that the loss of hydrogen bonds following amino acid changes can only explain a part of the observed blue-shift in the B850 band. The coupling of excitonic states to charge-transfer states, which is different in the two forms, contributes with a similar amount to the overall blue-shift.

Correction to: Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates.

In the original publication, under the subtitle Recovery: fluorescence recovery protein (FRP), paragraph 4 the text section enclosed in quotation marks does not occur in one of the original publications cited (Sluchanko et al. 2017a, b).

Energy transfer in purple bacterial photosynthetic units from cells grown in various light intensities.

Three photosynthetic membranes, called intra-cytoplasmic membranes (ICMs), from wild-type and the ∆pucBAabce mutant of the purple phototrophic bacterium Rps. palustris were investigated using optical spectroscopy. The ICMs contain identical light-harvesting complex 1-reaction centers (LH1-RC) but have various spectral forms of light-harvesting complex 2 (LH2). Spectroscopic studies involving steady-state absorption, fluorescence, and femtosecond time-resolved absorption at room temperature and at 77 K focused on inter-protein excitation energy transfer. The studies investigated how energy transfer is affected by altered spectral features of the LH2 complexes as those develop under growth at different light conditions. The study shows that LH1 â†’ LH2 excitation energy transfer is strongly affected if the LH2 complex alters its spectroscopic signature. The LH1 â†’ LH2 excitation energy transfer rate modeled with the Förster mechanism and kinetic simulations of transient absorption of the ICMs demonstrated that the transfer rate will be 2-3 times larger for ICMs accumulating LH2 complexes with the classical B800-850 spectral signature (grown in high light) compared to the ICMs from the same strain grown in low light. For the ICMs from the ∆pucBAabce mutant, in which the B850 band of the LH2 complex is blue-shifted and almost degenerate with the B800 band, the LH1 â†’ LH2 excitation energy transfer was not observed nor predicted by calculations.

Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates.

Cyanobacteria exhibit a novel form of non-photochemical quenching (NPQ) at the level of the phycobilisome. NPQ is a process that protects photosystem II (PSII) from possible highlight-induced photo-damage. Although significant advancement has been made in understanding the NPQ, there are still some missing details. This critical review focuses on how the orange carotenoid protein (OCP) and its partner fluorescence recovery protein (FRP) control the extent of quenching. What is and what is not known about the NPQ is discussed under four subtitles; where does exactly the site of quenching lie? (site), how is the quenching being triggered? (trigger), molecular mechanism of quenching (quenching) and recovery from quenching. Finally, a recent working model of NPQ, consistent with recent findings, is been described.

An improved crystal structure of C-phycoerythrin from the marine cyanobacterium Phormidium sp. A09DM.

C-Phycoerythrin (PE) from Phormidium sp. A09DM has been crystallized using different conditions and its structure determined to atomic resolution (1.14 Å). In order for the pigment present, phycoerythrobilin (PEB), to function as an efficient light-harvesting molecule it must be held rigidly (Kupka and Scheer in Biochim Biophys Acta 1777:94-103, 2008) and, moreover, the different PEB molecules in PE must be arranged, relative to each other, so as to promote efficient energy transfer between them. This improved structure has allowed us to define in great detail the structure of the PEBs and their binding sites. These precise structural details will facilitate theoretical calculations of each PEB's spectroscopic properties. It was possible, however, to suggest a model for which chromophores contribute to the different regions of absorption spectrum and propose a tentative scheme for energy transfer. We show that some subtle differences in one of these PEB binding sites in two of the 12 subunits are caused by crystal contacts between neighboring hexamers in the crystal lattice. This explains some of the differences seen in previous lower resolution structures determined at two different pH values (Kumar et al. in Photosyn Res 129:17-28, 2016).

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires.

A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified with nickel-chelating nitrilo-triacetic acid (Ni-NTA). The optical properties of conjugated nanostructures were studied using wide-field and confocal fluorescence microscopy. Plasmonic enhancement of RCs conjugated to AgNWs was observed as their fluorescence intensity dependence on the excitation wavelength does not follow the excitation spectrum of RC complexes in solution. The strongest effect of plasmonic interactions on the emission intensity of RCs coincides with the absorption spectrum of AgNWs and is observed for excitation into the carotenoid absorption. From the absence of fluorescence decay shortening, we attribute the emission enhancement to increase of absorption in RC complexes.