Rubisco proton production can drive the elevation of CO2 within condensates and carboxysomes.

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3-, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3- accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3- accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.

Reliable transition properties from excited-state mean-field calculations.

Delta-self-consistent field (ΔSCF) theory is a conceptually simple and computationally inexpensive method for finding excited states. Using the maximum overlap method to guide optimization of the excited state, ΔSCF has been shown to predict excitation energies with a level of accuracy that is competitive with, and sometimes better than, that of time-dependent density functional theory. Here, we benchmark ΔSCF on a larger set of molecules than has previously been considered, and, in particular, we examine the performance of ΔSCF in predicting transition dipole moments, the essential quantity for spectral intensities. A potential downfall for ΔSCF transition dipoles is origin dependence induced by the nonorthogonality of ΔSCF ground and excited states. We propose and test a simple correction for this problem, based on symmetric orthogonalization of the states, and demonstrate its use on bacteriochlorophyll structures sampled from the photosynthetic antenna in purple bacteria.

Understanding the role of the shrimp gut microbiome in health and disease.

With rapid increases in the global shrimp aquaculture sector, a focus on animal health during production becomes ever more important. Animal productivity is intimately linked to health, and the gut microbiome is becoming increasingly recognised as an important driver of cultivation success. The microbes that colonise the gut, commonly referred to as the gut microbiota or the gut microbiome, interact with their host and contribute to a number of key host processes, including digestion and immunity. Gut microbiome manipulation therefore represents an attractive proposition for aquaculture and has been suggested as a possible alternative to the use of broad-spectrum antibiotics in the management of disease, which is a major limitation of growth in this sector. Microbiota supplementation has also demonstrated positive effects on growth and survival of several different commercial species, including shrimp. Development of appropriate gut supplements, however, requires prior knowledge of the host microbiome. Little is known about the gut microbiota of the aquatic invertebrates, but penaeid shrimp are perhaps more studied than most. Here, we review current knowledge of information reported on the shrimp gut microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of Proteobacteria within this community. We discuss involvement of the microbiome in the regulation of shrimp health and disease and describe how the gut microbiota changes with the introduction of several economically important shrimp pathogens. Finally, we explore evidence of microbiome supplementation and consider its role in the future of penaeid shrimp production.

Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems.

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Purple bacteria photo-bioelectrochemistry: enthralling challenges and opportunities.

Purple non-sulfur bacteria are anoxygenic photosynthetic microorganisms characterized by an extremely versatile metabolism, allowing them to grow in a broad variety of conditions as well as in the presence of different contaminants. This characteristic motivates the interest in their employment in photo-bioelectrochemical systems applicable in environments with dynamic physico-chemical properties. While the photochemistry of purple bacteria has been intensively studied, their photo-bioelectrochemistry and extracellular electron transfer process with an electrode surface remain largely unexplored. Herein, the process of harvesting electrons from intact purple bacteria is reviewed, and the perspective of enthralling future research possibilities is presented, placing emphasis on the major challenges in the photo-bioelectrochemistry of purple bacteria.

Response of Wild Spotted Wing Drosophila (Drosophila suzukii) to Microbial Volatiles.

The olfactory cues used by various animals to detect and identify food items often include volatile organic compounds (VOCs) produced by food-associated microorganisms. Microbial VOCs have potential as lures to trap animal pests, including insect crop pests. This study investigated microorganisms whose VOCs are attractive to natural populations of the spotted wing drosophila (SWD), an invasive insect pest of ripening fruits. The microorganisms readily cultured from wild SWD and SWD-infested fruits included yeasts, especially Hanseniaspora species, and various bacteria, including Proteobacteria (especially Acetobacteraceae and Enterobacteriaceae) and Actinobacteria. Traps in a raspberry planting that were baited with cultures of Hanseniaspora uvarum, H. opuntiae and the commercial lure Scentry trapped relatively high numbers of both SWD and non-target drosophilids. The VOCs associated with these baits were dominated by ethyl acetate and, for yeasts, other esters. By contrast, Gluconobacter species (Acetobacteraceae), whose VOCs were dominated by acetic acid and acetoin and lacked detectable ethyl acetate, trapped 60-75% fewer SWD but with very high selectivity for SWD. VOCs of two other taxa tested, the yeast Pichia sp. and Curtobacterium sp. (Actinobacteria), trapped very few SWD or other insects. Our demonstration of among-microbial variation in VOCs and their attractiveness to SWD and non-pest insects under field conditions provides the basis for improved design of lures for SWD management. Further research is required to establish how different microbial VOC profiles may function as reliable cues of habitat suitability for fly feeding and oviposition, and how this variation maps onto among-insect species differences in habitat preference.

Selective Removal of B800 Bacteriochlorophyll a from Light-Harvesting Complex 2 of the Purple Photosynthetic Bacterium Phaeospirillum molischianum.

The selective removal of B800 bacteriochlorophyll (BChl) a from light-harvesting complex 2 (LH2) in purple photosynthetic bacteria is a clue about elucidation of the mechanism for the transfer of energy from these pigments to B850 BChl a and their roles in the LH2 protein structure. We demonstrated that the kinetics of the removal of B800 BChl a from two representative LH2 proteins derived from Phaeospirillum molischianum and Rhodoblastus acidophilus differed significantly, in contrast to the calculated binding enthalpy. These results may be interpreted as changes in the local structure near B800 BChl a with respect to the geometries of the original crystal structures upon removal of B800 BChl a. Despite the difficulty of removing B800 BChl a from molischianum-LH2, we prepared the molischianum-LH2 protein lacking B800 BChl a by combination of two detergents, n-dodecyl ß-d-maltoside and n-octyl ß-d-glucoside, under acidic conditions. Spectral and atomic force microscopy analyses indicated that the absence of B800 BChl a had little effect on the local structure in the vicinity of B850 BChl a and the circular arrangement in this protein. These results suggest that the hydrophobic domain near B850 BChl a is rigid and plays a major role in the structural formation of molischianum-LH2.

Teixobactin as a scaffold for unlimited new antimicrobial peptides: SAR study.

It looks that a new era of antimicrobial peptides (AMPs) started with the discovery of teixobactin, which is a "head to side-chain" cyclodepsipeptide. It was isolated from a soil gram-negative b-proteobacteria by means of a revolutionary technique. Since there, several groups have developed synthetic strategies for efficient synthesis of this peptide and its analogues as well. Herein, all chemistries reported as well as the biological activity of the analogues are analyzed. Finally, some inputs regarding new trends for the next generation of analogues are discussed.

Coordination of the biliverdin D-ring in bacteriophytochromes.

Phytochrome proteins translate light into biochemical signals in plants, fungi and microorganisms. Light cues are absorbed by a bilin chromophore, leading to an isomerization and a rotation of the D-ring. This relays the signal to the protein matrix. A set of amino acids, which is conserved across the phytochrome superfamily, holds the chromophore in the binding pocket. However, the functional role of many of these amino acids is not yet understood. Here, we investigate the hydrogen bonding network which surrounds the D-ring of the chromophore in the resting (Pr) state. We use UV/vis spectroscopy, infrared absorption spectroscopy and X-ray crystallography to compare the photosensory domains from Deinococcus radiodurans, the phytochrome 1 from Stigmatella aurantiaca, and a D. radiodurans H290T mutant. In the latter two, an otherwise conserved histidine next to the D-ring is replaced by a threonine. Our infrared absorption data indicate that the carbonyl of the D-ring is more strongly coordinated by hydrogen bonds when the histidine is missing. This is in apparent contrast with the crystal structure of the PAS-GAF domain of phytochrome 1 from S. aurantiaca (pdb code 4RPW), which did not resolve any obvious binding partners for the D-ring carbonyl. We present a new crystal structure of the H290T mutant of the PAS-GAF from D. radiodurans phytochrome. The 1.4 Å-resolution structure reveals additional water molecules, which fill the void created by the mutation. Two of the waters are significantly disordered, suggesting that flexibility might be important for the photoconversion. Finally, we report a spectral analysis which quantitatively explains why the histidine-less phytochromes do not reach equal Pfr-type absorption in the photoequilibrium compared to the Deinococcus radiodurans wild-type protein. The study highlights the importance of water molecules and the hydrogen bonding network around the chromophore for controlling the isomerization reaction and spectral properties of phytochromes.

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

Microaerophilic Fe(II)-Oxidizing Zetaproteobacteria Isolated from Low-Fe Marine Coastal Sediments: Physiology and Composition of Their Twisted Stalks.

Microaerophilic Fe(II) oxidizers are commonly found in habitats containing elevated Fe(II) and low O2 concentrations and often produce characteristic Fe mineral structures, so-called twisted stalks or tubular sheaths. Isolates originating from freshwater habitats are all members of the Betaproteobacteria, while isolates from marine habitats belong almost exclusively to the Zetaproteobacteria So far, only a few isolates of marine microaerophilic Fe(II) oxidizers have been described, all of which are obligate microaerophilic Fe(II) oxidizers and have been thought to be restricted to Fe-rich systems. Here, we present two new isolates of marine microaerophilic Fe(II)-oxidizing Zetaproteobacteria that originate from typical coastal marine sediments containing only low Fe concentrations (2 to 11 mg of total Fe/g of sediment [dry weight]; 70 to 100 µM dissolved Fe2+ in the porewater). The two novel Zetaproteobacteria share characteristic physiological properties of the Zetaproteobacteria group, even though they come from low-Fe environments: the isolates are obligate microaerophilic Fe(II) oxidizers and, like most isolated Zetaproteobacteria, they produce twisted stalks. We found a low organic carbon content in the stalks (∼0.3 wt%), with mostly polysaccharides and saturated aliphatic chains (most likely lipids). The Fe minerals in the stalks were identified as lepidocrocite and possibly ferrihydrite. Immobilization experiments with Ni2+ showed that the stalks can function as a sink for trace metals. Our findings show that obligate microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria group are not restricted to Fe-rich environments but can also be found in low-Fe marine environments, which increases their overall importance for the global biogeochemical Fe cycle.IMPORTANCE So far, only a few isolates of benthic marine microaerophilic Fe(II) oxidizers belonging to the Zetaproteobacteria exist, and most isolates were obtained from habitats containing elevated Fe concentrations. Consequently, it was thought that these microorganisms are important mainly in habitats with high Fe concentrations. The two novel isolates of Zetaproteobacteria that are presented in the present study were isolated from typical coastal marine sediments that do not contain elevated Fe concentrations. This increases the knowledge about possible habitats in which Zetaproteobacteria can exist. Furthermore, we show that the physiology and the typical organo-mineral structures (twisted stalks) that are produced by the isolates do not notably differ from the physiology and the cell-mineral structures of isolates from environments with high Fe concentrations. We also showed that the organo-mineral structures can function as a sink for trace metals.

Evaluating Nitrogen-Containing Biosynthetic Products Produced by Saltwater Culturing of Several California Littoral Zone Gram-Negative Bacteria.

The biosynthetic potential of marine-sediment-derived Gram-negative bacteria is poorly understood. Sampling of California near-shore marine environments afforded isolation of numerous Gram-negative bacteria in the Proteobacteria and Bacteriodetes phyla, which were grown in the laboratory to provide extracts whose metabolites were identified by comparative analyses of LC-mass spectrometry and MSn data. Overall, we developed an assemblage of seven bacterial strains grown in five different media types designed to coax out unique secondary metabolite production as a function of varying culture conditions. The changes in metabolite production patterns were tracked using the GNPS MS2 fragmentation pattern analysis tool. A variety of nitrogen-rich metabolites were visualized from the different strains grown in different media, and strikingly, all of the strains examined produced the same new, proton-atom-deficient compound, 1-methyl-4-methylthio-ß-carboline (1), C13H12N2S. Scale-up liquid culture of Achromobacter spanius (order: Burkholderiales; class: Betaproteobacteria) provided material for the final structure elucidation. The methods successfully combined in this work should stimulate future studies of molecules from marine-derived Gram-negative bacteria.

How Markovian is exciton dynamics in purple bacteria?

We investigate the extent to which the dynamics of excitons in the light-harvesting complex LH2 of purple bacteria can be described using a Markovian approximation. To analyse the degree of non-Markovianity in these systems, we introduce a measure based on fitting Lindblad dynamics, as well as employing a recently introduced trace-distance measure. We apply these measures to a chromophore-dimer model of exciton dynamics and use the hierarchical equation-of-motion method to take into account the broad, low-frequency phonon bath. With a smooth phonon bath, small amounts of non-Markovianity are present according to the trace-distance measure, but the dynamics is poorly described by a Lindblad master equation unless the excitonic dimer coupling strength is modified. Inclusion of underdamped, high-frequency modes leads to significant deviations from Markovian evolution in both measures. In particular, we find that modes that are nearly resonant with gaps in the excitonic spectrum produce dynamics that deviate most strongly from the Lindblad approximation, despite the trace distance measuring larger amounts of non-Markovianity for higher frequency modes. Overall we find that the detailed structure in the high-frequency region of the spectral density has a significant impact on the nature of the dynamics of excitons.

Investigating the Biosynthesis of Natural Products from Marine Proteobacteria: A Survey of Molecules and Strategies.

The phylum proteobacteria contains a wide array of Gram-negative marine bacteria. With recent advances in genomic sequencing, genome analysis, and analytical chemistry techniques, a whole host of information is being revealed about the primary and secondary metabolism of marine proteobacteria. This has led to the discovery of a growing number of medically relevant natural products, including novel leads for the treatment of multidrug-resistant Staphylococcus aureus (MRSA) and cancer. Of equal interest, marine proteobacteria produce natural products whose structure and biosynthetic mechanisms differ from those of their terrestrial and actinobacterial counterparts. Notable features of secondary metabolites produced by marine proteobacteria include halogenation, sulfur-containing heterocycles, non-ribosomal peptides, and polyketides with unusual biosynthetic logic. As advances are made in the technology associated with functional genomics, such as computational sequence analysis, targeted DNA manipulation, and heterologous expression, it has become easier to probe the mechanisms for natural product biosynthesis. This review will focus on genomics driven approaches to understanding the biosynthetic mechanisms for natural products produced by marine proteobacteria.

N-Acyl Dehydrotyrosines, Tyrosinase Inhibitors from the Marine Bacterium Thalassotalea sp. PP2-459.

Thalassotalic acids A-C and thalassotalamides A and B are new N-acyl dehydrotyrosine derivatives produced by Thalassotalea sp. PP2-459, a Gram-negative bacterium isolated from a marine bivalve aquaculture facility. The structures were elucidated via a combination of spectroscopic analyses emphasizing two-dimensional NMR and high-resolution mass spectrometric data. Thalassotalic acid A (1) displays in vitro inhibition of the enzyme tyrosinase with an IC50 value (130 µM) that compares favorably to the commercially used control compounds kojic acid (46 µM) and arbutin (100 µM). These are the first natural products reported from a bacterium belonging to the genus Thalassotalea.

Biological denitrification using poly(butanediol succinate) as electron donor.

Poly(butanediol succinate) (PBS), a biodegradable polymer, was used as both solid carbon source and biofilm carrier for biological nitrate removal process, in which PBS was filled in a packed-bed bioreactor. The denitrification performance and the microbial diversity of biofilm attached on the surface of PBS were investigated. The experimental results showed that the volumetric denitrification rate was 0.60 kg m(-3) day(-1) when NO3-N loading rate was 0.63 kg m(-3) day(-1), and the average NO2-N concentration was below 0.20 mg L(-1). The effluent pH value decreased slightly from a range of 6.98-7.87 to 6.46-7.18. The analysis of microbial community structure of biofilm by pyrosequencing method showed that Proteobacteria was the most abundant phylum (89.87 %), and ß-Proteobacteria represented the most abundant class. Among the 76 identified genera, Dechloromonas (10.26 %), Alicycliphilus (9.15 %), Azospira (8.92 %), and Sinobacteraceae-uncultured (8.75 %) were the abundant genera. PBS, as a promising alternative carbon source, is a suitable solid carbon source and biofilm carrier for nitrate removal.

3-Acetyl-Chlorophyll Formation in Light-Harvesting Complexes of Purple Bacteria by Chemical Oxidation.

Dedicated to the memory of Yuriy Nikolayevich Kozlov Oxidation of bacteriochlorophyll (BChl) with potassium ferricyanide in membranes and LH2 complexes (carotenoid-less and control samples) from the purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides as well as BChl photobleaching in a model system have been studied. The oxidation of BChl depended on the type of bacteria. BChl850 was rapidly oxidized in samples from Alc. minutissimum, and BChl800 and BChl850 were slowly oxidized in samples from Rb. sphaeroides. The carotenoids were not involved in protecting BChl from chemical oxidation in the light-harvesting complexes. The appearance of BChl oxidation product was registered in the absorption spectra (absorption maximum about 700 nm) and by HPLC analysis. The oxidized BChl was identified as 3-acetyl-chlorophyll. It differed from BChl by the presence of a double bond in pyrrole ring II at the 7-8 position. The extinction coefficient of 3-acetyl-chlorophyll was about 10 times less than that of BChl850 in the LH2 complex from Alc. minutissimum. In the BChl → 3-acetyl-chlorophyll transition, the binding constant of the latter with LH2 complex as compared with that of BChl did not change dramatically, as indicated by: (i) preserved electrophoretic mobility of the complex; (ii) the presence of 3-acetyl-chlorophyll in the complex after separation; (iii) the presence of a 3-acetyl-chlorophyll CD signal that was proportional to its absorption spectrum.

Proteorhodopsin Activation Is Modulated by Dynamic Changes in Internal Hydration.

Proteorhodopsin, a member of the microbial rhodopsin family, is a seven-transmembrane α-helical protein that functions as a light-driven proton pump. Understanding the proton-pumping mechanism of proteorhodopsin requires intimate knowledge of the proton transfer pathway via complex hydrogen-bonding networks formed by amino acid residues and internal water molecules. Here we conducted a series of microsecond time scale molecular dynamics simulations on both the dark state and the initial photoactivated state of blue proteorhodopsin to reveal the structural basis for proton transfer with respect to protein internal hydration. A complex series of dynamic hydrogen-bonding networks involving water molecules exists, facilitated by water channels and hydration sites within proteorhodopsin. High levels of hydration were discovered at each proton transfer site-the retinal binding pocket and proton uptake and release sites-underscoring the critical participation of water molecules in the proton-pumping mechanism. Water-bridged interactions and local water channels were also observed and can potentially mediate long-distance proton transfer between each site. The most significant phenomenon is after isomerization of retinal, an increase in water flux occurs that connects the proton release group, a conserved arginine residue, and the retinal binding pocket. Our results provide a detailed description of the internal hydration of the early photointermediates in the proteorhodopsin photocycle under alkaline pH conditions. These results lay the fundamental groundwork for understanding the intimate role that hydration plays in the structure-function relationship underlying the proteorhodopsin proton-pumping mechanism, as well as providing context for the relationship of hydration in proteorhodopsin to other microbial retinal proteins.

Channelrhodopsins: a bioinformatics perspective.

Channelrhodopsins are microbial-type rhodopsins that function as light-gated cation channels. Understanding how the detailed architecture of the protein governs its dynamics and specificity for ions is important, because it has the potential to assist in designing site-directed channelrhodopsin mutants for specific neurobiology applications. Here we use bioinformatics methods to derive accurate alignments of channelrhodopsin sequences, assess the sequence conservation patterns and find conserved motifs in channelrhodopsins, and use homology modeling to construct three-dimensional structural models of channelrhodopsins. The analyses reveal that helices C and D of channelrhodopsins contain Cys, Ser, and Thr groups that can engage in both intra- and inter-helical hydrogen bonds. We propose that these polar groups participate in inter-helical hydrogen-bonding clusters important for the protein conformational dynamics and for the local water interactions. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Proteorhodopsin.

Proteorhodopsins are the most abundant retinal based photoreceptors and their phototrophic function might be relevant in marine ecosystems. Here, we describe their remarkable molecular properties with a special focus on the green absorbing variant. Its distinct features include a high pKa value of the primary proton acceptor stabilized through an interaction with a conserved histidine, a long-range interaction between the cytoplasmic EF loop and the chromophore entailing a particular mode of color tuning and a variable proton pumping vectoriality with complex voltage-dependence. The proteorhodopsin family represents a profound example for structure-function relationships. Especially the development of a biophysical understanding of green proteorhodopsin is an excellent example for the unique opportunities offered by a combined approach of advanced spectroscopic and electrophysiological methods. This article is part of a Special Issue entitled: Retinal Proteins-You can teach an old dog new tricks.